Gd3-mimetic peptides

ABSTRACT

GD3-Mimetic peptides which contain an amino acid sequence represented by any of SEQ ID NOS: 1 to 4 or an amino acid sequence derived therefrom by substitution, deletion, addition, or insertion of one or more amino acid residues and attaining specific binding to an anti-GD3 antibody; and medicinal compositions containing the same.

TECHNICAL FIELD

The present invention relates to GD3-mimetic peptides, and moreparticularly to novel peptides which are considered to structurallymimic GD3, which are known as tumor associated antigens found in tumorssuch as melanoma.

BACKGROUND ART

GD3 is one form of sialosyl sphingoglycolipids; G stands for ganglioside(sialosyl sphingoglycolipid) and D stands for disialo. GD3, like othertumor-associated antigens such as GM2, GM3, GD2, and GT3, is known to beexpressed on tumor cells such as those of human melanoma. The structuralformula of GD3 is represented by NeuAc α2-8 NeuAc α2-3 Gal β1-4 Glc β1-1Cer.

Previous reports regarding GD3 have revealed, among other findings, thatdevelopment of a tumor correlates to expression of GD3, that GD3 ishighly expressed on melanoma cells, and that administration of mouseanti-GD3 monoclonal antibody suppresses growth of tumors of melanomapatients. On the basis of these findings, GD3 has become of keeninterest in immunotherapy of these types of tumors.

Thus, human immune response against GD3 is expected to providebeneficial effects during the clinical course of pathologicalconditions; in fact, a variety of clinical tests have been performed inthe technical field of vaccines. However, successful results thatfulfill the above expectation have yet not been reported (see, forexample, Cheresh, D. A., et al., Proc. Natl. Acad. Sci., USA., 81,5767-5771 (1984): Herlyn, M., et al., Cancer Res., 45, 5670-5676 (1985):Houghton, A. N., et al., Proc. Natl. Acad. Sci., USA., 82, 1242-1246(1985): Livingston, P. O., Immunological Rev., 145, 147-163 (1995):Livingston, P. O., et al., Cancer Immunol. Immunother., 45, 1-9 (1997)).

Gangliosides, which have been found to be associated with tumors, arethus known to serve as a useful target in the immunological attackagainst cancer. However, they are acknowledged to have poorimmunogenicity.

In order to overcome this drawback, there has been developed anddisclosed a cancer vaccine composition for inducing or stimulatingimmune response of antibodies against gangliosides (Japanese PatentApplication Laid-Open (kokai) No. 8-53366). This composition is anN-glycosylated product of ganglioside (i.e., N-glycosyl GM3).

Similarly, U.S. Pat. No. 5,102,663 discloses a 9-O-acetylated product ofganglioside (9-O-acetylated GD3)

Moreover, Japanese Kohyo Publication No. 8-508978 discloses that asimilar cancer vaccine, GD3 complex vaccine (GD3-keyhole limpethemocyanin complex), exhibits significantly improved antibody responses.According to the disclosure of this publication, a double bond in theceramide backbone of GD3 are cleaved with ozone for chemicalmodification, thus introducing an aldehyde group, and the aldehyde groupis caused to bind to the aminolysyl group of protein through reductiveamination, to thereby construct a complex with a synthesizedmulti-antigenic peptide displaying repeats of malarial T cell epitopes,coat protein of Neisseria meningitidis (OMP), cationized bovine serumalbumin (cBSA), keyhole limpet hemocyanin (KLH) and polylysin. Thatpublication also describes that the most effective immunologicaladjuvant is QS-21, obtained through extraction of the bark of a treefound in South America and called Quillajasaponaria Molina (AquilaPharmaceuticals, Worcester, Mass., U.S.A.: Kensil, C. R. et al., J.Immunol., 146, 431 (1991)).

Conventional vaccines prepared through employment of GD3 per se as anantigen exhibit only weak immune responses and their effects aretransient. Moreover, they have drawbacks, in that the raw material GD3is not readily available. That is, generally speaking, mass productionof a desired ganglioside from a living organism is very difficult. Also,synthesis of a ganglioside through chemical synthesis or geneticengineering is very difficult.

When vaccines are prepared through a variety of chemical modificationsof GD3, particularly in chemical treatment performed for improvingantigenicity or in preparation of complexes, disadvantages areencountered in terms of intricate procedure in relation to procurement,preparation, synthesis, etc. of raw materials for antigens andcomplexes, and necessity for selection of immunoadjuvants.

Meanwhile, in recent years, molecular biological techniques have beenemployed in the technical fields of complex saccharides, and techniquesfor replacing the sugar chain with peptide are now under development.Previously, the present inventors have successfully obtained, from aphage display random peptide library through biopanning using amonoclonal antibody against a sugar chain, a peptide which exhibitsspecific binding to an antibody against one form of ganglyoside, GD1α.

This peptide (15 mer) has been found to mimic the sugar chain structureof a complex glycolipid (and thus is called a glyco-replica peptide), tobe bound, with specificity to, a monoclonal antibody against glycolipidGD1α, and to inhibit binding of an antibody to antigen GD1α.

The present inventors have also produced this peptide through chemicalsynthesis, and have confirmed that the peptide reacts with a monoclonalantibody for GD1α, that a (chemically synthesized) replica peptide ofGD1α inhibits cell adhesion of cancer cells of highly metastatic cancercell lines, and that the replica peptide inhibits metastasis of cancercells (Japanese Patent Application Laid-Open (kokai) No. 10-237099).

In addition, the present inventors have succeeded in obtaining, from arandom peptide library, a peptide which exhibits to modulate glycosidaseactivity and reacts with specificity with an antibody againstlactotetraosyl ceramide or lactoneotetrasyl ceramide (see JapanesePatent Application Laid-Open (kokai) No. 10-237098 and Saibo Kogaku, DaiIshikawa and Takao Taki, 16 (12) 1821-1828 (1997)).

A recently published report discloses study results similar to theabove-described ones obtained by the present inventors. Specifically,Qiu, J., et al. have disclosed in Hybridoma 18(1) 103-112 (1999) that a15-16 mer peptide containing a Trp-Arg-Tyr sequence and obtained from aphage display peptide library through use of an antibody against aGD2/GD3 antigen exhibits cross reaction with the antibody.

Willers, J., et al. describe that they have obtained, from two phagedisplay peptide libraries of phages displaying 15-mer and 8-merpeptides, four phage-displayed peptides capable of binding to anti-GD3monoclonal antibodies MB3.6, MG22, and MG21 (Peptides, 20, 1021-1026(1999)). According to this publication, these peptides were found toexhibit ability to binding to the anti-GD3 antibody employed forselection, and this binding ability was inhibited by GD3, but desiredimmunogenicity was not observed for any of the peptides.

An object of the present invention is to provide a novel peptide whichmimics the structure of the sugar chain of ganglioside GD3 and exhibitshigh affinity with anti-GD3 antibody.

Another object of the present invention is to provide an immunogenicpeptide capable of producing GD3-specific antibody; in particular, apeptide having a characteristic feature such that an antibody producedthrough immunization with an immunogen comprising the peptidecross-reacts with GD3, and therefore, has utility as a vaccine whichreplaces GD3.

A still further object of the present invention is to provide a DNAsequence coding for the above-mentioned peptide, a recombinantexpression vector in which the sequence has been integrated, a host cellharboring the vector, and a recombinant expression product produced bythe cell.

A still further object of the present invention is to provide apharmaceutical composition containing as an active ingredient theabove-mentioned peptide or the recombinant expression vector.

DISCLOSURE OF THE INVENTION

The present inventors have carried out extensive studies, and have founda novel amino acid sequence exhibiting high affinity to an anti-GD3antibody. The inventors have also found that an antiserum obtainedthrough immunization with a GD3-mimetic peptide comprising a peptidecontaining the amino acid sequence exhibits cross-reactivity with GD3,and that the GD3-mimetic peptide finds utility as a vaccine thatreplaces GD3. The present invention has been accomplished on the basisof these findings.

Accordingly, the present invention provides a GD3-mimetic peptidecontaining an amino acid sequence represented by any one of SEQ ID NOs:1 to 4 or an amino acid sequence derived therefrom by substitution,deletion, addition, or insertion of one or more amino acid residues andexhibiting binding specificity to an anti-GD3 antibody.

In particular, the present invention provides a GD3-mimetic peptidewhich is in a fused form of peptide—fused with a carrier protein capableof enhancing immunogenicity—and the GD3-mimetic peptide, wherein thecarrier protein is keyhole limpet hemocyanin; a multi-antigenicGD3-mimetic peptide containing at least one species of the GD3-mimeticpeptide; an immunogenic GD3-mimetic peptide having the ability toproduce a GD3-specific antibody; and a GD3-mimetic peptide having anamino acid sequence represented by any one of SEQ ID NOS: 1 to 4.

The present invention also provides a pharmaceutical compositioncontaining, as an active ingredient, the GD3-mimetic peptide.

The present invention encompasses, among others, the followingembodiments:

(1) a DNA sequence encoding the GD3-mimetic peptide of the presentinvention, particularly a DNA sequence encoding an amino acid sequencerepresented by any one of SEQ ID NOS: 1 to 4, preferably an amino acidsequence represented by SEQ ID NO: 3 or 4;

(2) a DNA sequence as described above, having a DNA sequence representedby any one of SEQ ID NOS: 5 to 8;

(3) a recombinant expression vector into which at least one of these DNAsequences has been inserted;

(4) a host cell into which the above recombinant expression vector hasbeen incorporated;

(5) a pharmaceutical composition containing, as an active ingredient,the recombinant expression vector;

(6) use of the pharmaceutical composition of the present invention as avaccine for stimulating induction of an antibody capable of recognizingGD3 or for enhancing production of the antibody;

(7) use of the pharmaceutical composition of the present invention as adrug for suppressing a tumor or cancer or for inhibiting cancerousmetastasis;

(8) use of the drug for treating GD3-expressing tumors or cancer,particularly pathological conditions selected from the group consistingof melanoma, cancer of the large intestine, ovarian cancer, livercancer, breast cancer, brain tumor, kidney cancer, pancreas cancer,cervical cancer, esophageal cancer, lung cancer, and stomach cancer; and

(9) a pharmaceutical composition which is a liposome drug preparation.

As used herein, abbreviations for amino acids, peptides, nucleotidesequences, nucleic acids, etc. are in accordance with the IUPACstandards; IUB standards; “Guidelines for Drafting Specifications orSimilar Materials Containing a Nucleotide Sequence or an Amino AcidSequence,” (edited by Japanese Patent Office); and symbols customarilyemployed in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing binding affinity, as determined through themethod described in relation to Example 2, of the immunogenic peptidesaccording to the present invention and anti-GD3 antibody.

FIG. 2 shows sequences of multi-antigenic peptides employed in Example5.

FIG. 3 is a graph showing binding affinity of GD3-mimetic peptides andanti-GD3 monoclonal antibody.

FIG. 4 is a graph showing inhibitory effect exerted by immobilizedGD3-mimetic peptides against binding between GD3 and 4F6 (anti-GD3antibody).

FIG. 5 is a graph showing inhibitory effect exerted by exogenously addedGD3-mimetic peptides against binding between GD3 and 4F6.

FIG. 6 is a graph showing inhibitory effect exerted by exogenously addedGD3-mimetic peptides (nine residues) against binding between GD3 and4F6.

FIG. 7 is a graph showing the relationship between dilution factor ofserum samples from mice immunized with peptide R4 and results of ELISAon GD3.

FIG. 8 is a graph showing the relationship between dilution factor ofserum samples from mice immunized with R4MAP for two months and resultsof ELISA on GD3.

FIG. 9 is a graph showing the relationship between dilution factor ofserum samples from mice immunized with R4MAP for two months and resultsof ELISA performed on R4MAP peptide.

BEST MODES FOR CARRYING OUT THE INVENTION

The GD3-mimetic peptides of the present invention will next be describedin detail.

The GD3-mimetic peptides of the present invention are characterized bycontaining an amino acid sequence represented by any one of SEQ ID NOS:1 to 4 or an amino acid sequence derived therefrom by substitution,deletion, addition, or insertion of one or more amino acid residues andbeing capable of binding to an antibody to GD3 (anti-GD3 antibody) withspecificity.

As used herein, “anti-GD3 antibody” is a term generally employed in theart and is defined as a specific antibody capable of recognizing GD3(i.e., binding to GD3). The antibody preferably exhibits no crossreactivity with other gangliosides relating to GD3 and is particularlypreferably a monoclonal antibody.

The amino acid sequence represented by any one of SEQ ID NOS: 1 to 4 ischaracterized by mimicking the sugar chain structure of GD3. Thus, apeptide containing the amino acid sequence, exhibiting specific bindingto an anti-GD3 antibody, is a preferred example of the GD3-mimeticpeptides of the present invention.

The aforementioned specific amino acid sequence may be an amino acidsequence derived through modification of a portion of amino acidresidues or a portion of the sequence, so long as the structuralfeature; i.e., mimicking the sugar chain structure of GD3, is maintainedor presented.

No particular limitation is imposed on the extent and position of themodification (i.e., substitution, deletion, addition, or insertion) ofthe amino acid sequence, so long as the modified amino acid sequence isan equivalent in terms of effect and exhibits characteristics similar tothose of the amino acid sequence represented by any one of SEQ ID NOS: 1to 4; i.e., mimicking the sugar chain structure of GD3, is maintained orpresented.

Modification may be generally effected to an extent of 80% or morehomology, preferably 90% or more homology.

Among the peptides falling within the scope of the present invention, apeptide having at least two cysteine residues; e.g., a peptide having anamino acid sequence represented by SEQ ID NO: 2, is considered tospontaneously cyclize. Such a cyclic peptide is active (exhibitsspecific binding to an anti-GD3 antibody) even when the peptide ispresent in a linear-chain form. Therefore, one or both cysteine residuesincluded in the peptide or an amino acid sequence portion sandwiched bythese two cysteine residues can be deleted without greatly affecting thestructural characteristics of mimicking the sugar chain structure ofGD3. This phenomenon is supported by the literature (e.g., Koivunen, etal., J. Biol. Chem., 268, 20205-20210 (1993)).

Examples of the aforementioned deleted amino acid sequence include asequence of nine amino acid residues (PHFDSLLYP) residing in theN-terminus of GD3R2.

Any of the GD3-mimetic peptides of the present invention is formed of apeptide containing one of the aforementioned specific amino acidsequences falling within the scope of the present invention. Thus, thestructural characteristic; i.e., mimicking the sugar chain structure ofGD3, is maintained or presented, and the GD3-mimetic peptide ischaracterized by specific binding to an anti-GD3 antibody.

From the viewpoint of immunogenicity and other properties, theaforementioned peptides containing any one of the specific amino acidsequences has a number of amino acid residues of at least 5, preferably8 to 60, more preferably 8 to 30, still more preferably 10 to 20.

The GD3-mimetic peptides of the present invention preferably have anamino acid sequence represented by SEQ ID NO: 3 or 4, particularlypreferably an amino acid sequence represented by SEQ ID NO: 4.Alternatively, the GD3-mimetic peptides preferably have an amino acidsequence derived from an amino acid sequence represented by SEQ ID NO: 3or 4 by substitution, deletion, addition, or insertion of one or moreamino acid residues.

More preferably, the GD3-mimetic peptides of the present invention areimmunogenic peptides which are capable of producing a GD3-specificantibody. In other words, the peptides can induce a GD3-specificantibody.

Specifically, the GD3-mimetic peptides of the present invention, whichare characterized by containing an amino acid sequence represented byany one of SEQ ID NOS: 1 to 4, per se have the ability to produce aGD3-specific antibody, and therefore, such peptides are preferred as theimmunogenic peptides of the present invention.

The immunogenic peptides of the present invention also encompasspeptides which become to have a desired immunogenicity throughtransformation into immunogenicity-enhanced forms, as well as peptidesof the immunogenicity-enhanced forms.

Thus, the immunogenic peptides having ability to produce a GD3-specificantibody, which peptides are preferred embodiments of the presentinvention, encompass the unmodified peptides per se andimmunogenicity-enhanced forms of the GD3-mimetic peptides such as a formof a peptide fused with a customary carrier protein for enhancingimmunogenicity and a form of a multi-antigen peptide, these two formsfalling within the scope of the present invention.

Whether the GD3-mimetic peptides of the present invention maintain orpresent the structural characteristic of mimicking the sugar chainstructure of GD3 (i.e., exhibit specific binding to an anti-GD3antibody) can be confirmed by checking reactivity with the anti-GD3antibody through a routine test method. In addition, whether theGD3-mimetic peptides of the present invention are immunogenic peptideshaving the ability to produce a GD3-specific antibody can be confirmedby checking, through a routine test method, cross reactivity of inducedantibodies with GD3.

Detection of binding specificity of the GD3-mimetic peptides of thepresent invention to an anti-GD3 monoclonal antibody and reactivity ofGD3 with an antibody induced by an immunogenic peptide will bespecifically described in the below-described Examples.

The GD3-mimetic peptides of the present invention encompass thefollowing embodiments.

(a) a GD3-mimetic peptide comprising a peptide containing an amino acidsequence formed through fusion or linkage of a plurality of members ofat least one species of the aforementioned specific amino acidsequences;

(b) a GD3-mimetic peptide in a multi-antigen peptide form comprising apeptide containing at least one species of the aforementioned specificamino acid sequence; and

(c) a GD3-mimetic peptide in a fusion peptide form, fused with a carrierprotein or a peptide which can enhance immunogenicity or promote immuneresponse.

These specific examples of the GD3-mimetic peptides of the presentinvention will next be described in more detail.

In the below-described Examples, peptides represented by GD3R1, GD3R2,GD3R3, and GD3R4 denote peptides having amino acid sequences representedby SEQ ID NOS: 1 to 4, respectively. These peptides are preferredexamples of the GD3-mimetic peptides of the present invention. Peptidesformed of arbitrarily continuous 9 to 14 amino acid residues containedin an amino acid sequence represented by any one of SEQ ID NOS: 1 to 4are also preferred. Of these, peptides formed of 9 to 14 N-terminus orC-terminus amino acid residues contained in an amino acid sequencerepresented by any one of SEQ ID NOS: 1 to 4 are more preferred.Peptides formed of nine N-terminus or C-terminus amino acid residuescontained in an amino acid sequence represented by any one of SEQ IDNOS: 1 to 4 are particularly preferred. As shown in Examples, thesepeptides were obtained by selecting peptides exhibiting specific bindingto an anti-GD3 antibody by use of phage display libraries.

The screening step is not necessarily repeated for producing theGD3-mimetic peptides of the present invention. If desired, screening isperformed, for example, in the following manner.

Specifically, a large population of molecules (library) are produced,and peptides of interest are identified through screening the moleculelibrary. A phage display library can be employed for screening. Themethod for producing the library and screening method are disclosed inthe literature (e.g., Scott, J. M. and Smith, G. P., Science, 249,386-390 (1990); Smith, G. P. and Scott, J. K., Methods in Enzymology,217, 228-257 (1993)). Examples of preferable methods for identifying theGD3-mimetic peptides of the present invention include methods foridentifying glycolipid sugar chain-mimetic peptides disclosed in, forexample, Japanese Patent Application Laid-Open (kokai) Nos. 10-237099and 10-237098, and Dai ISHIKAWA and Takao TAKI, Cell Engineering, 16(12) 1821-1828 (1997).

Phage clones which display a peptide of interest can be screened andselected through a binding (to an antibody) test by use of an antibodyrecognizing GD3, preferably a monoclonal antibody having highspecificity to GD3.

By determining DNA sequences of the selected phage clones, a GD3-mimeticpeptide of interest can be identified. The DNA sequences can be readilydetermined through any method known in the art; e.g., a dideoxy method[Proc. Natl. Acad. Sci. USA., 74, 5463-5467 (1977)] or the Maxam-Gilbertmethod [Method in Enzymology, 65, 499 (1980)]. A commercial sequence kitor similar means may be conveniently employed to determine thecorresponding nucleotide sequences.

(Production of the GD3-Mimetic Peptides of the Present Invention)

The GD3-mimetic peptides of the present invention can be produced inaccordance with their amino acid sequences through any customarychemical synthesis method, such as a typical liquid-phase or solid-phasepeptide synthesis methods.

More specifically, the peptide synthesis methods include a stepwiseelongation method in which respective amino acids are successivelylinked in accordance with amino acid sequence information, to therebyextend the chain; and a fragment condensation method in which fragmentsare preliminary synthesized from several amino acids and the fragmentsare coupled. The GD3-mimetic peptides of the present invention can besynthesized through either method.

Any condensation method can be employed for the above peptide synthesis.Examples thereof include an azide method, a mixed acid anhydride method,a DCC method, an active ester method, a redox method, a DPPA(diphenylphosphorylazide) method, a DCC+additive (e.g.,1-hydroxybenzotriazole, N-hydroxysuccinamide, orN-hydroxy-5-norbornene-2,3-dicarboximide) method, and the Woodwardmethod.

The solvent employable in these methods is appropriately selected fromcustomary solvents well-known to be usable in a variety of peptidecondensation reactions. Examples include dimethylformamide (DMF),dimethyl sulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran(THF), ethyl acetate, and mixtures thereof.

Upon the above peptide synthesis reaction, carboxyl groups of the aminoacids and peptides whose groups are not involved in the reaction may begenerally protected through esterification to thereby form a lower alkylester (e.g., methyl ester, ethyl ester, or tert-butyl ester); or anaralkyl ester (e.g., benzyl ester, p-methoxybenzyl ester, orp-nitrobenzyl ester). A side-chain functional group of an amino acid;e.g., the hydroxyl group of Tyr, may be protected by a group such asacetyl, benzyl, benzyloxycarbonyl, or tert-butyl, but the protection isnot essential. The guanidino group of Arg may be protected by anappropriate protective group such as nitro, tosyl,2-methoxybenzenesulfonyl, methanesulfonyl, benzyloxycarbonyl,isobornyloxycarbonyl, or adamantyloxycarbonyl.

Deprotection reaction of the protective groups of amino acids, peptides,and the finally obtained immunogenic peptides of the present inventionhaving the above protective groups may be performed through anycustomarily employed method. For example, there may be performedcatalytic reduction and chemical deprotection by use of an agent such asliquid ammonia/sodium, hydrogen fluoride, hydrogen bromide, hydrogenchloride, trifluoroacetic acid, acetic acid, formic acid, ormethanesulfonic acid.

Purification of the thus-obtained GD3-mimetic peptides of the presentinvention can be appropriately performed through a routine method suchas an ion-exchange resin method, partition chromatography, gelchromatography, affinity chromatography, high-performance liquidchromatography (HPLC), or counter current distribution, which arecustomarily employed in the field of peptide chemistry.

Alternatively, the GD3-mimetic peptides of the present invention can beproduced through a genetic engineering technique employing the DNAsequences according to the present invention encoding the peptides.

The above procedure is carried out through a routine method. Forexample, synthesis of DNA, production of an expression vector which canexpress the DNA, and the method of expressing the vector in host cellsmay be carried out in accordance with generally employed geneticengineering techniques (See Molecular Cloning 2d. Ed., Cold SpringHarbor Lab. Press (1989); Zoku-Seikagaku Jikken Koza “Gene study I, II,and III,” edited by The Japanese Biochemical Society (1986); etc.).

The DNA encoding a GD3-mimetic peptide of the present invention may beprepared through a routine method on the basis of the amino acidsequence information of the GD3-mimetic peptide provided by the presentinvention (e.g., Science, 224, 1431 (1984); and Biochem. Biophys. Res.Comm., 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990 (1983)).

More specifically, the DNA can be chemically synthesized through thephosphoramidite method or the triester method. The synthesis can beperformed by use of a commercially available automated oligonucleotidesynthesizer. Double-strand fragments can be produced from asingle-strand product yielded through chemical synthesis includingsynthesis of complementary strands and annealing of the strands underappropriate conditions; or by addition of complementary strands with anappropriate primer sequence by use of a DNA polymerase.

If desired, the encoding amino acid sequence of the aforementioned DNAmay be modified through any known method, such as site-specificvariation introduction employing an oligonucleotide (Zoller, M., et al.,Nucl. Acids Res., 10, 6487-6500 (1982)), or cassette mutagenesis (Well,J., et al., Gene, 34, 315-323 (1985)).

Production and expression of a peptide of interest by use of the DNA maybe carried out through any method known in the art (e.g., Science, 224,1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); and Proc.Natl. Acad. Sci. USA., 80, 5990 (1983)). Production and expression offused peptides and proteins may be performed in accordance with a methodof Ohno et al. “Protein Experiment Protocol 1 function analysis,separate vol. of Cell Engineering, Experiment Protocol Series (1997),Shujun-sha) or other methods.

The thus-obtained peptides of interest can be isolated and purifiedthrough a variety of separation procedures on the basis of physical,chemical, and other properties (e.g., “Biochemistry Data Book II,”1175-1259, 1st edition, 1st issue, Jun. 23, 1980, Tokyo Kagaku Dojin;Biochemistry, 25 (25), 8274-8277 (1986); and Eur. J. Biochem., 163,313-321 (1987)). Examples of separation procedures include generalreconstruction treatment; treatment by use of a protein-precipitatingagent (salting out); centrifugation; osmotic shock procedure; ultrasoniccrushing; ultrafiltration; liquid chromatography techniques such asmolecular sieve chromatography (gel filtration), adsorptionchromatography, ion-exchange chromatography, affinity chromatography,and high-performance liquid chromatography (HPLC); dialysis; andcombinations thereof.

The GD3-mimetic peptides of the present invention are more preferablyimmunogenic peptides having the ability to produce a GD3-specificantibody, which may be an immunogenicity-enhanced form such as a fusionpeptide form, peptide fused with a carrier protein, for enhancingimmunogenicity, or a multi-antigen peptide form.

The GD3-mimetic peptides of the present invention in fusion form, fusedwith a carrier protein for enhancing immunogenicity, are obtained bybinding any one of the peptides according to the present invention to acustomary carrier protein for enhancing immunogenicity.

No particular limitation is imposed on the type of the carrier protein,so long as the protein can enhance immunogenicity, and a variety ofproteins and peptides which impart higher immunogenicity on the basis ofthe carrier effect or which promote immune response of living organismsmay be employed as the carrier protein. The carrier protein may be aprotein or peptide which additionally exerts a pharmaceutical effectsuch as anti-tumor activity.

When the GD3-mimetic peptides of the present invention are used asdrugs, the carrier protein is selected from pharmaceutically acceptableproteins and peptides. Examples of preferable carrier proteins includekeyhole limpet hemocyanin (KLH) and cytokines such as IL-12 and GM-CSF.Examples of the aforementioned proteins and peptides which exert apharmaceutical effect include IFN-α, IFN-β, IFN-γ, IL-1, IL-2, TNF,TGF-β, angiostatin, thrombospondin, and endostatin.

Binding of the above peptide and the carrier protein may be carried outthrough the aforementioned peptide synthesis method.

The binding may also be carried out by use of the DNA or genes thereofthrough the aforementioned recombinant technique.

Thus, GD3-mimetic peptides of the present invention in fused form can beobtained.

The GD3-mimetic peptides of the present invention may be multi-antigenpeptides (MAP). These peptides are characterized in that a plurality ofthe GD3-mimetic peptides according to the present invention aredisplayed on a base molecule.

The MAP form of the GD3-mimetic peptides of the present invention can bepreferably produced by use of a dendrimer serving as a base molecule ora skeleton.

As is generally known, a dendrimer is a molecule (e.g., sphericalmolecule) of a dendritic or star-like configuration, which molecule hasbranches (repeating units) having a plurality of functional groups (see,e.g., Japanese Kohyo Patent Publication No. 60-500295; Japanese PatentApplication Laid-Open (kokai) Nos. 63-99233 and 3-263431; U.S. Pat. Nos.4,507,466 and 4,568,737; Polymer Journal, 17, p. 117 (1985); AngewandteChem. Int. Engl., 29, 138-175 (1990); and Macromolecules, 25, p. 3247(1992)).

No particular limitation is imposed on the dendrimer employed in thepresent invention, and the dendrimer may be formed of a core serving asa polymerization-initiating site; an internal layer (generation)comprising repeating units connected to the initiation core; and anouter surface formed of functional groups connected to the branches.

Characteristics such as dimensions, morphology, and reactivity of thedendrimer can be regulated by modifying the initiation core, the numberof generations, and the type of repeating units employed in eachgeneration. Specifically, the dimensions of a dendrimer can be readilyincreased by increasing the number of generations to be included (see,e.g., U.S. Pat. No. 4,694,064).

Examples of typical MAP forms of the GD3-mimetic peptides of the presentinvention include a dendrimer comprising a nitrogen atom serving as aninitiation core site; a —CH₂CH₂CONHCH₂CH₂— fragment serving as arepeating unit (branch) connecting to each core site; and a plurality ofGD3-mimetic peptides connected to the outermost terminals of thedendrimer branches; and a dendrimer comprising an amino acid (e.g., Lys,Arg, Glu, or Asp) serving as an initiation core site, the same aminoacid serving as a repeating unit directly connected to each core site,and a plurality of GD3-mimetic peptides connected to terminals of thebranches in a similar manner.

The aforementioned dendrimer having a nitrogen atom serving as aninitiation core site can be produced through a customary method orobtained as a commercial product (Polysciences, Inc., 400 Vally Road,Warrington, Pa., 18976 U.S.A.). The aforementioned dendrimer having anamino acid serving as an initiation core site and the same amino acidserving as a repeating unit directly connected to each initiation coresite can be produced through the aforementioned peptide synthesismethod. In addition, commercial products such asFmoc₈-Lys₄-Lys₂-Lys-βAla-Alko resin (product of Watanabe Kagaku Kogyo)may also be used.

More specifically, the aforementioned dendrimers can be produced throughcondensation of α,ω-diamino acids serving as repeating units and a resinfor synthesizing a solid-phase peptide, where two amino groups of eachamino acid have been protected by protective groups, which are identicalto or different from each other, in the presence or absence of a spacer;and repetition of removal of the protective groups.

Resins typically employed for peptide synthesis can be employed as theresin for synthesizing a solid-phase peptide. Examples includepolystyrene resin, polyacrylamide resin, polystyrene-polyethylene glycolresin, the terminal groups of these resins being capped with afunctional group such as chloromethyl, 4-(hydroxymethyl)phenoxy, or4-((α-2′,4′-dimethoxyphenyl)-9-fluorenylmethoxycarbonylaminomethyl)phenoxy.One or more amino acids may be employed as spacers.

Examples of α,ω-diamino acids include lysine, ornithine,1,4-diaminobutyric acid, and 1,3-diaminopropionic acid. Removal of theprotective groups can be performed through the aforementioned peptidesynthesis method. Examples of functional groups include an amino group,a carboxyl group, and a hydroxyl group.

The number of branch layers is adjusted to 2n by performing condensationof a repeating unit and removal of protective groups n times in total.Specifically, the number may fall within a range of 2 to 16.

By binding the GD3-mimetic peptides according to the present inventionto the functional groups connected to terminals of the branches of thedendrimer, an MAP of interest can be obtained. The procedure can beperformed in accordance with the aforementioned peptide synthesismethod.

The thus-produced MAP can be purified through chromatography or asimilar method in a routine manner by use of a resin which attains sizeexclusion in a matrix such as Sephacryl S-300 (product of Pharmacia).

The GD3-mimetic peptides according to the present invention which arecaused to be bound to the terminal of branches of the MAP according tothe present invention are not necessarily identical with one another,and different species may be combined arbitrarily. Examples ofcombinations of different GD3-mimetic peptides include a combination ofSEQ ID NOS: 1, 3, and 4; and a combination of a peptide having an aminoacid sequence of 15 amino acid residues shown in any one of SEQ ID NOS:1 to 4 and a peptide having an amino acid sequence of nine amino acidresidues shown in FIG. 2. Such a complex MAP enhances stability thereofin the blood and tissues of the target to which the MAP is administered,and enhances immunogenicity or other properties of the molecule to whichthe MAP has been bound. Thus, production of a GD3 antibody by any of theGD3-mimetic peptides of the present invention may be promoted.

Any of the MAPs of the present invention may be transformed into acomplex MAP in which the aforementioned carrier protein (e.g.polypeptide promoting immune response such as IL-12 or GM-CSF) forenhancing immunogenicity is bound as a portion of the GD3-mimeticpeptide of the present invention or to an initiation core site. Othergangliosides which are expressed on tumor cells other than theGD3-mimetic peptides of the present invention, such as one or moremimetic peptides (e.g., GM2, GM3, GD1, GD2, GD3, and GT3), can also beemployed as MAP constituents in combination with any of the GD3-mimeticpeptides of the present invention. Examples of complex MAPs include acombination of at least one peptide containing an amino acid sequencerepresented by any one of SEQ ID NOS: 1 to 4 or an amino acid sequenceshown in FIG. 2 with a replica peptide mimicking GD1α disclosed in theaforementioned Japanese Patent Application Laid-Open (kokai) No.10-237099.

The thus-produced MAP forms of the GD3-mimetic peptides of the presentinvention exhibit excellent immunogenicity and, therefore, can exertdesired effects; i.e., inducing production of an anti-GD3 antibody orincreasing production of the antibody.

The GD3-mimetic peptides of the present invention in MAP form exerteffects as desired for a vaccine; i.e., a carcinostatic effect and aninhibitory effect for cancerous metastasis. In addition, an arbitrarydrug such as a drug promoting immune response is incorporated into theMAPs and the thus-modified MAPs can be administered. Thus, MAPs areadvantageous in that induction of a target antibody can be promoted,production of an antibody can be further increased, and other effectscan be enhanced.

(Pharmaceutical Composition of the Present Invention)

The present invention provides a pharmaceutical composition for humanand animals containing, as an active ingredient, any of the GD3-mimeticpeptides of the present invention.

The pharmaceutical composition is useful as, among others, acancer-diagnostic agent on the basis of the effect that the activeingredient binds to an antibody to a cancer-related GD3 antigen.

Preferably, the pharmaceutical composition of the present inventioncontains, as an active ingredient, any of the GD3-mimetic peptides ofthe present invention in the form of an immunogenic peptide having theability to produce a GD3-specific antibody.

The GD3-mimetic peptides of the present invention in the form of theabove immunogenic peptide mimic the structure of GD3 and exhibit animmunogenicity similar to that of GD3. Thus, the peptides exhibitanti-tumor effect on the basis of activation of cytoxic effectsdepending on a complement or an induced or produced antibody oractivation of cytoxic T cells; and an intercellular adhesion inhibitoryeffect by mediation of GD3 in tumor cells expressing GD3. The peptidesare useful for a variety of pharmaceutical uses.

Examples of use of the drugs provided by the pharmaceutical compositioninclude a vaccine for stimulating induction of an antibody recognizingGD3 or enhancing production of the antibody; anti-tumor use,carcinostatic use, or inhibitory use for cancerous metastasis; andtreatment for GD3-expressing tumors or cancer, particularly diseasesselected from the group consisting of melanoma, cancer of largeintestine, ovarian cancer, liver cancer, breast cancer, brain cancer,kidney cancer, pancreas cancer, cervical cancer, esophageal cancer, lungcancer, and stomach cancer.

The pharmaceutical composition of the present invention can be preparedas a composition containing an pharmaceutically effective amount of anyof the GD3-mimetic peptides of the present invention and apharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers employed in the presentinvention are known in the art and are appropriately selected inaccordance with the form of the composition to be prepared. When thecomposition to be prepared is an aqueous solution, carriers such aswater and physiological buffers can be used without limitation, andglycol, glycerol, and injectable organic esters such as olive oil mayalso be used.

The pharmaceutical composition of the present invention may furthercontain another active ingredient and an arbitrary ingredient forstabilizing absorption of the active ingredient and enhancingabsorption. Examples of arbitrary ingredients include hydrocarbons suchas glucose, sucrose, and dextran; antioxidants such as ascorbic acid andglutathione; chelating agents; stabilizers such as low-molecular-weightproteins and albumin; and vehicles.

Into the pharmaceutical composition of the present invention, arbitraryadditives for designing drug preparations can be appropriatelyincorporated. Examples include a variety of generally employed additivessuch as antiseptic agents, tonicity agents, buffers, stabilizers,solubilizers, and absorption promoters. Examples of the antisepticagents include those effective for fungi and bacteria such asbenzalkonium chloride, benzethonium chloride, chlorhexidine, parabens(e.g., methyl parben, ethyl paraben), and thimerosal. Examples oftonicity agents include polyhydric alcohols such as D-mannitol,D-sorbitol, D-xylitol, glycerin, glucose, maltose, sucrose, andpropylene glycol; and electrolytes such as sodium chloride. Examples ofstabilizers include tocopherol, butylhydroxyanisole,butylhydroxytoluene, ethylenediaminetetraacetic acid salts (EDTA), andcysteine.

One form of the pharmaceutical composition of the present invention is aliposome preparation. This preparation will next be described in detail.The preparation can be produced by causing any of the GD3-mimeticpeptides of the present invention to be held on liposome comprising, asa membrane-forming ingredient, acidic phospholipid or comprising, asmembrane-forming ingredients, neutral phospholipid and acidicphospholipid.

No particular limitation is imposed on the type of neutral phospholipidand acidic phospholipid serving as membrane-forming ingredients, andlipid species which are customarily used in such liposome preparationsmay be used singly or in combination of two or more species.

Membrane of the liposome is formed through a customary method by use ofacidic phospholipid as a membrane-forming ingredient or by use ofneutral phospholipid and acidic phospholipid in combination asmembrane-forming ingredients. The acidic phospholipid content is about0.1 to about 100 mol % based on the total amount of liposomemembrane-forming ingredients, preferably about 1 to about 90 mol %, morepreferably about 10 to about 50 mol %.

Upon preparation of the liposome membrane, additives such as cholesterolmay be incorporated into phospholipid, to thereby control flowability,thus facilitating preparation of liposome membrane. The amount ofcholesterol incorporated into phospholipid is generally equivalent tothat of phospholipid or less, preferably 0.5-1 equivalent.

The proportion of acidic phospholipid to active ingredient(s) containedin a liposome dispersion is about 0.5 to about 100 equivalents,preferably about 1 to about 60 equivalents, more preferably about 1.5 toabout 20 equivalents. The amount of any of the GD3-mimetic peptides ofthe present invention is some mol % to some tens of mol % based on thetotal lipid species, preferably about 5 to about 10 mol %, with about 5mol % being typically employed.

Preparation, concentration, particle size control, and other processesof the liposome can be performed through a customary method. Theaforementioned various additives may be incorporated into the liposomepreparation in accordance with needs.

Upon production of liposome, fatty acid (e.g., behenic acid, stearicacid, palmitic acid, myristic acid, or oleic acid), an alkyl group, acholesteryl group, or a similar group may be bound to any of theGD3-mimetic peptides of the present invention. Production of a liposomepreparation by use of such a modified peptide can be performed through acustomary method (e.g., Long Circulating Liposomes: Old drugs, Newtherapeutics., M. C. Woodle, G. Storm, Eds: Springer-Verlag Berlin(1998)).

No particular limitation is imposed on the amount of activeingredient(s) contained in the pharmaceutical composition (drugpreparation) of the present invention, and the amount can be selectedfrom a wide range, so long as it is pharmaceutically effective.

Generally, any of the GD3-mimetic peptides of the present invention iscontained in a drug preparation in an amount of about 0.00001 to about70 wt. %, preferably about 0.0001 to about 5 wt. %. No particularlimitation is imposed on the amount of administration of the drugpreparation, and the amount is appropriately selected from a wide rangein accordance with desired therapeutic effects, the method (way) ofadministration, the therapy period, and the age, sex, and otherconditions of patients. Generally, the daily dose per patient per bodyweight (kg) preferably falls within a range of about 0.01 μg to about 10mg as reduced to active ingredient(s), preferably about 0.1 μg to about1 mg. The drug preparation may be administered once per day or in adivided manner.

The pharmaceutical composition of the present invention is preferablyemployed as the aforementioned vaccine composition. In use as a vaccinecomposition, the composition of the present invention is preferablyadministered along with a pharmaceutically effective amount of anadjuvant in combination so as to enhance its anti-tumor effect.

No particular limitation is imposed on the type of the adjuvant, andexamples include Freund's complete adjuvant, muramyl dipeptide, BCG,IL-12, N-acetylmuramin-L-alanyl-D-isoglutamine (MDP), thymosin α1, andQS-21. Although the amount of adjuvant to be administered is limiteddepending on a type of pathological conditions of human and animalscaused, after administration, by immune reaction such as malacia, pain,and erythema of the skin; fever; headache; or muscle pains, the dailydose of adjuvant per patient per body weight (kg) is generally about 0.1μg to about 1,000 μg, preferably about 1 μg to some hundreds of μg.

The pharmaceutical composition of the present invention can be used incombination with other drugs, such as an immune-response-promotingpeptide and a cancer chemotherapy agent (anti-cancer agent). The amountof the drug administered in combination is appropriately determined inaccordance with the pharmaceutically effective amount of the drug. Forexample, when GM-CSF is used, the daily dose thereof per body weight(kg) of the patient is generally about 0.1 μg to about 1,000 μg,preferably about 1 μg to some hundreds of μg.

Examples of the aforementioned drugs used in combination include cancerchemotherapy agents including 5-fluorouracil (5-FU), such as alkylatingagents, metabolism antagonists, anti-tumor antibiotic preparations,anti-tumor vegetable-originating preparations; and the aforementionedcytokines having an anti-tumor activity.

Upon combination use, a drug can be incorporated into MAP formGD3-mimetic peptides of the present invention in the aforementionedmanner. In addition, a drug delivery substance such as a microdevicehaving a microchamber which can accommodate other drugs and whichaccommodates the GD3-mimetic peptides of the present invention can alsobe used. Examples of such drug delivery substances include biologicalsubstances such as liposome, microcapsules having permeable orsemi-permeable membranes, and other microdevices having microchambers.These substances are non-toxic and may be biodegradable.

Binding of these drug delivery substances and the GD3-mimetic peptide ofthe present invention can be preformed through a customary method (e.g.,Harlow and Lane, Antibodies: A laboratory manual, Cold Spring HarborLab. Press (1988); and Hermanson, Bioconjugate Techniques, AcademicPress (1996)). A pharmaceutical composition containing a substance suchas a cytokine containing a carcinostatic and exerting anti-tumoractivity is produced through a known method (e.g., Liposomal applicationto cancer therapy, Y. Namba, N. Oku, J., Bioact. Compat. Polymers, 8,158-177 (1993)).

When the pharmaceutical composition of the present invention is employedas a diagnostic agent, any of the GD3-mimetic peptides of the presentinvention—the active ingredient—may be labeled for its detection.Labeling can be performed through a customary method, and materials suchas radioactive compounds, fluorescent substances, enzymes, biotin, andcontrast media can be used.

Through employment of such a diagnostic agent, an anti-GD3 antibodycontained in a variety of samples such as cancer tissues and cells andbody fluids such as blood can be detected. Thus, the diagnostic agent isuseful for cancer diagnosis, monitoring of pathological conditions, etc.

(DNA of the Present Invention)

The present invention provides DNA comprising a sequence encoding any ofthe GD3-mimetic peptides of the present invention. The DNA is useful inthe aforementioned production of the GD3-mimetic peptides of the presentinvention through a genetic engineering technique. The DNA is suitablyused for producing a DNA vaccine containing the DNA as an activeingredient.

As mentioned above, the DNA encoding the GD3-mimetic peptides of thepresent invention is preferably DNA encoding immunogenic peptides havingthe ability to produce a GD3-specific antibody which immunogenicpeptides are embodiments of the GD3-mimetic peptides of the presentinvention. Furthermore, the DNA may encode the aforementionedparticularly preferred forms of the GD3-mimetic peptides of the presentinvention.

The aforementioned vaccine comprises a pharmaceutical compositioncontaining, as an active ingredient, the DNA encoding immunogenicpeptides having the ability to produce a GD3-specific antibody whichimmunogenic peptides are embodiments of the GD3-mimetic peptides of thepresent invention or a recombinant expression vector which can expressthe DNA.

The pharmaceutical composition is useful for a DNA vaccine targetingcancer cells or tissues of mammals including human, and uses of thepharmaceutical composition are similar to those described in relation tothe pharmaceutical composition containing, as an active ingredient, anyof the GD3-mimetic peptides of the present invention.

The aforementioned DNA vaccine can be prepared as a pharmaceuticalcomposition though a customary method by use of a pharmaceuticallyacceptable carrier. Preferably, the vaccine contains a physiologicallyacceptable solution such as sterilized physiological saline orsterilized buffered physiological saline. Similar to the case of theaforementioned pharmaceutical composition containing, as an activeingredient, any of the GD3-mimetic peptides of the present invention,the composition may be a liposome drug preparation and may be used incombination with an adjuvant or a similar material.

The pharmaceutical composition may contain an arbitrary drug andadditives. Examples of the drug include a drug such as calcium ionspromoting incorporation of DNA into cells, and examples of the additivesinclude materials for facilitating transfection, such as theaforementioned liposome, fluorocarbon emulsions, cochleates, tubules,gold particles, biodegradable microspheres, and cationic polymers.

The amount of DNA which can be expressed and is introduced to avaccination host or the amount of transcripted RNA which is introducedto a vaccination host is selected from a wide range of amount ofadministration. The amount is varied in accordance with, for example,the capacity of employed transcription and translation promoters. Theintensity of immune response varies in accordance with the level ofexpression of protein and the immunogenicity of expressed gene products.Generally, the vaccine is parenterally administered in an effectiveamount of about 1 ng to about 5 mg based on DNA, preferably about 100 ngto about 2.5 mg, more preferably about 1 μg to about 750 μg, still morepreferably about 10 μg to 300 μg. Generally, the vaccine is administereddirectly to muscle tissues. Alternatively, there may be also employedother administration methods such as hypodermic injection, introductionto the corium, skin impression, intraperitoneal administration,intravenous administration, and inhalation.

In general, the vaccine is administered not once, but boostervaccination is effected one or more times so as to enhance the effect ofthe vaccine while conditions of the administered specimens aremonitored. Alternatively, vaccination of DNA may be followed by boosterby use of the aforementioned pharmaceutical composition comprising theGD3-mimetic peptide of the present invention. In addition, theaforementioned various combinations may enhance the therapy efficacy.

No particular limitation is imposed on the type of recombinantexpression vector which can express the DNA of the aforementioned DNAvaccine, and expression vectors generally employed in such types of DNAvaccines and other employable expression vectors can be used. Thesevectors can be produced through a customary method.

(Antibody of the Present Invention)

The GD3-mimetic peptides of the present invention function as antigensand produce the corresponding antibodies. Specifically, any of thepeptides which binds to GD3 binds to cells such as malignant tumor cells(e.g., melanoma cells) which express GD3, thereby producing an antibody(neutralizing antibody) which inhibits proliferation of the cells andexhibits activity to inhibit metastasis of the cells. The presentinvention also provides such an antibody.

Confirmation of production of such an antibody is regarded as a test fordiagnosing that the GD3-mimetic peptide of the present invention is animmunogenic peptide having the ability to produce a GD3-specificantibody.

The antibodies according to the present invention include monoclonal andpolyclonal antibodies. These antibodies can be produced through acustomarily employed technique by use of the GD3-mimetic peptide as animmunogen.

Next, production of the monoclonal antibody will be described in detail.The monoclonal antibody can be produced, for example, by fusing plasmacells (immunocytes) of a mammal which has been immunized with the aboveimmunogen and plasmacytoma cells (myeloma cells) of the mammal, tothereby produce fused cells (hybridomas); selecting a clone producing adesired antibody recognizing GD3 (monoclonal antibody); and culturingthe clone. The monoclonal antibody is fundamentally produced inaccordance with a routine method (e.g., Hanfland, P., Chem. Phys.Lipids, 15, 105 (1975); Hanfland, P., Chem. Phys. Lipids, 10, 201(1976); and Koscielak, J., Eur. J. Biochem., 37, 214 (1978)).

No particular limitation is imposed on the mammal which is immunizedwith the immunogen in the above method, and mice, rats, etc. arepreferably used from the viewpoint of compatibility to plasmacytomacells to be fused. Immunization can be performed through a customarymethod such as administration of the above immunogen to a mammal throughinjection (e.g., intravenous, intradermal, hypodermal, orintraperitoneal).

More specifically, when mice are used, preferably, the immunogen isdiluted to an appropriate concentration with a diluent such asphysiological phosphate buffered saline (PBS) or physiological salineand is administered, in combination with an optional customary adjuvantin accordance with needs, to an animal specimen several times atintervals of 2 to 14 days in a total amount of about 100 to about 500μg/mouse. Examples of the adjuvant which is preferably employed in theabove process include a pertussis vaccine, Freund's complete adjuvant,and ALUM. Preferably employed immunocytes include spleen cells whichhave been extirpated about three days after final administration of theimmunogen.

A variety of known mammal plasmacytoma cells can be employed as counterparent cells to be fused with the above immunocytes. The fusion can beperformed through any known method, such as the method described inMilstein et al. (Method in Enzymology, 73, 3 (1981)). Isolation andcloning of the yielded hybridomas can be performed through customarymethods.

The target antibody-producing strains can be retrieved through a varietyof methods generally employed for detecting antibodies (“HybridomaMethod and Monoclonal Antibody,” issued by R&D Planning, 30-53, Mar. 5,1982). Examples of the methods include the ELISA method (Engvall, E.,Meth. Enzymol., 70, 419-439 (1980)), a plaque method, a spot method, anagglutination method, an ochterlony method, and radioimmunoassay (RIA).Upon retrieval, the aforementioned immunogen and GD3 can be employed.

The thus-yielded hybridomas producing a desired monoclonal antibodyrecognizing GD3 can be subcultured in a customary medium and preservedfor a long period of time in liquid nitrogen. Examples of methods forcollecting a monoclonal antibody from the hybridomas include a methodcomprising culturing the hybridomas in a routine manner and collectingan antibody from a culture supernatant; and a method comprisingadministering the hydridomas to a mammal compatible to the hybridomasfor proliferating the hybridomas and collecting an antibody from theascites. The former method is suitable for producing a high-purityantibody, whereas the latter method is suitable for mass production ofan antibody.

The culture supernatant and mouse's ascites obtained through the abovemethods and containing antibody-producing hybridomas may be employed ascrude antibody liquids. Alternatively, the crude liquids may be purifiedthrough a routine method; i.e., method such as ammonium sulfatefractionation, salting out, gel filtration, ion-exchange chromatography,or affinity chromatography such as protein A column chromatography, tothereby yield a purified antibody.

EXAMPLES

The present invention will next be described in more detail by way ofexamples, which should not be construed as limiting the inventionthereto.

The following abbreviations are employed herein.

TBS: Tris phosphate-buffered salineTC: tetracyclineKM: kanamycinPEG/NaCl: polyethylene glycol/sodium chlorideTFA: trifluoroacetic acid

Example 1 Identification of GD3-Mimetic Peptide (1) Preparation of PhageDisplay Libraries

According to the procedure disclosed by Nishi T., Saya H., et al. (FEBSLetter, 399, 237-240 (1996)), random DNA fragments encoding peptides ofrandom 15 amino acid residues were inserted into phage coat protein pIIIgene, to thereby prepare phage display libraries of interest containinggenes capable of expressing peptides of random 15 amino acid residues onthe coat surface of respective phages.

Since the phages contain a TC resistant gene, E. coli infected with thephages is TC resistant. The phage libraries thus constructed were storedin a TBS solution containing 0.02% NaN₃.

Characteristic features of the above-mentioned phage display librariesare described by Scott, J. K. and Smith G. P. (Science, 249, 386-390(1990)).

(2) Immobilization of Anti-GD3 Monoclonal Antibody

The anti-GD3 antibody employed is an anti-GD3 monoclonal antibody 4F6(hereinafter referred to as GD3 antibody 4F6). The GD3 antibody 4F6 is amouse monoclonal antibody (IgG) against GD3 (Thomas C. P., et al.,Glycoconj. J., 13 (3), 377-384 (1996)), which was kindly provided by Dr.Jacques Portoukalian (INSERM, France).

A recombinant protein A Sepharose suspension (50 μL) (rProtein ASepharose Fast Flow; product of Pharmacia Biotech K.K., Code No:17-1279-01, Lot No: 237393) was transferred to a 0.5-mL Eppendorf tube,and TBS (400 μL) was added thereto. Subsequently, GD3 antibody 4F6 (1mL) was added, and the mixture was allowed to react overnight at 4° C.under thorough stirring. After completion of reaction, the mixture wascentrifuged at 3,000 rpm for 5 minutes, the supernatant was discarded,TBS (0.5 mL) was added to the residue, and the mixture was thoroughlystirred. The resultant mixture was transferred to a 1.5-mL Eppendorftube. TBS (0.5 mL) was added thereto, the mixture was centrifuged at3,000 rpm for 5 minutes, and the supernatant was discarded.Subsequently, TBS (1 mL) was added to the residue, the mixture wascentrifuged at 3,000 rpm for 5 minutes, and the supernatant was removed.TBS (100 μL) was added to the residue, to thereby yield a suspension;i.e., a suspension of recombinant protein A Sepharose (100 μL) bound toGD3 antibody 4F6, which was stored at 4° C. until use.

An aliquot of the thus-obtained recombinant protein A Sepharosesuspension (50 μL) was transferred to a 1.5-mL Eppendorf tube, and TBS(900 μL) was added thereto. Subsequently, 12 mg/mL mouse immunoglobulinstandard serum (Code No: RS10-101-2; product of Bethyl; purchased fromCosmo Bio Co., Ltd.) in TBS (total volume of the resultant solution: 100μL) was added, and the mixture was allowed to react overnight at 4° C.under thorough stirring. (Protein A was reacted with an excessive amountof IgG so as to minimize the amount of protein A remaining unreacted.)After completion of reaction, the mixture was centrifuged at 3,000 rpmfor 5 minutes, the supernatant was removed, TBS (1 mL) was added to theresidue, and the mixture was thoroughly stirred. The resultant mixturewas transferred to a 1.5-mL Eppendorf tube. The mixture was centrifugedat 3,000 rpm for 5 minutes, and the supernatant was removed. After thisprocedure was repeated three times, TBS (100 μL) was added to theresidue, to thereby prepare a suspension; i.e., a suspension of arecombinant protein A Sepharose (100 μL) bound to mouse IgG (250 μg),which was stored at 4° C. until use.

(3) Preparation of E. coli

E. coli employed as a host of phage is E. coli K91KAN (kanamycinresistant strain; kindly provided by Professor SAYA, Hideyuki,Department of Tumor Genetics and Biology of Kumammoto University).

By use of a disposable platinum loop, E. coli K91KAN was inoculated intoan NZY plate containing 100 μg/mL kanamycin (product of Wako PureChemicals Industries, Ltd.) for incubation at 37° C. overnight. On thefollowing day, the plate was removed from the incubator and stored at 4°C. until use.

The day before the E. coli was infected with phages, a small amount ofthe E. coli which had been stored at 4° C. in the plate was collected byuse of a platinum loop, and inoculated into an NZY medium (5 mL)containing 100 μg/mL kanamycin, followed by shaking culture at 200 rpmat 37° C. overnight (pre-culture). On the following day, the culturedmixture (100 μL) was transferred to a fresh NZY medium (10 mL), followedby shaking culture at 37° C. for 4 hours. Thereafter, in order for thecells to develop F-cilia, shaking was stopped, and the cells were leftto stand for 30 minutes. The thus-prepared E. coli cells were infectedwith phages as described below.

The aforementioned NZY medium was obtained through the following steps:NZ amine A (product of Wako Pure Chemicals Industries, Ltd.; Code No:541-00241) (10 g), brewer's yeast extraction (trade name: EBIOS, productof Asahi Breweries, Ltd.) (5 g), and NaCl (5 g) were dissolved indistilled water (1 L); 5N NaOH (1 mL) was added thereto; pH of themixture was adjusted to 7.5; the mixture was sterilized in an autoclave;and the resultant mixture was stored at room temperature until use.

(4) Amplification of Phage

The E. coli host cells were infected with the phages prepared in theprocedure described in (1).

Briefly, the above-prepared E. Coli K91KAN (10 μL) and a diluted phagepreparation (10 μL) were placed in a 15-mL centrifuge tube, and themixture was allowed to react at room temperature for 15 minutes. An NZYmedium (containing 0.2 μg/mL TC) (1 mL) which had been heated in advanceto 37° C. was added to the mixture, followed by shaking culture at 2,000rpm at 37° C. for 40 minutes. An aliquot (200 μL) of the resultantmixture was inoculated into an NZY plate (containing 20 μg/mL TC and 100μg/mL KM) for incubation at 37° C. overnight. On the following day, thenumber of colonies was counted.

A negative control was prepared by adding E. coli K91KAN (10 μL) to thedilution liquid (10 μL). Since this mixture remained sensitive to TC,amplification of phages did not occur in the TC-containing NZY plate.

Amplification of phages that were recovered through biopanning, whichwill be described herein later, was performed in the following manner.

The entire amount of the phage solution, excepting the amount (2 μL) tobe used for titer measurement, was placed in a 1.5-mL Eppendorf tube,and the above-prepared E. coli K91KAN (100 μL) was added thereto,followed by reaction at room temperature for 15 minutes. Aftercompletion of reaction, the entire amount of the mixture was added to aTC-supplemented NZY medium (20 mL; the amount of TC: 0.2 μg/mL) whichhad been heated in advance to 37° C. in a 50-mL centrifuge tube,followed by shaking culture at 200 rpm at 37° C. for 40 minutes. 20mg/mL TC (20 μL) was added, and the mixture was again subjected toshaking culture at 37° C. overnight. On the following day, the resultantmixture was centrifuged at 3,000 rpm for 10 minutes. The supernatant wastransferred to an Oakridge centrifuge tube and centrifuged at 12,000 rpmfor 10 minutes, to thereby remove E. coli cells completely. Thesupernatant was transferred to another Oakridge centrifuge tube, andPEG/NaCl (3 mL) was added thereto, followed by thorough stirring. Theresultant mixture was left to stand at 4° C. for 4 hours or more.

Subsequently, centrifugation was performed at 12,000 rpm for 10 minutesfor causing sedimentation of amplified phages. The supernatant wasremoved, and the phage sediment was suspended in TBS (1 mL). Thesuspension was transferred to a 1.5-mL Eppendorf tube and centrifuged at15,000 rpm for 10 minutes, to thereby remove insoluble substances. Thesupernatant was transferred to another Eppendorf tube, PEG/NaCl (150 μL)was added thereto, and the mixture was thoroughly stirred and left tostand at 4° C. for 1 hour or more.

The mixture was centrifuged at 15,000 rpm for 10 minutes, to therebyallow the phages to precipitate. The supernatant was removed, and theprecipitated phage pellet was resuspended in TBS (200 μL) containing0.02% NaN₃. The suspension was centrifuged at 15,000 rpm for 10 minutes,to thereby settle insoluble substances. The sediment was transferred toa 500-μL Eppendorf tube for storage at 4° C., and employed as the sourceof “amplified phage” in each round of panning.

The phages were titered in the following manner.

For titration of phages recovered in each round, 10²-, 10³-, and10⁴-fold diluted phage preparations were used, whereas for titration ofamplified phages, 10⁷-, 10⁸-, and 10⁹-fold diluted preparations wereused. TBS/gelatin (product of Wako Pure Chemicals Industries, Ltd.) wasemployed as a dilution solution, and dilution was performed inaccordance with the following dilution scheme.

×10² dilution: phage solution (2 μL)+TBS/gelatin (198 μL)×10³ dilution: 10²-fold diluted phage solution (10 μL)+TBS/gelatin (90μL)×10⁴ dilution: 10²-fold diluted phage solution (2 μL)+TBS/gelatin (198μL)×10⁶ dilution: 10⁴-fold diluted phage solution (2 μL)+TBS/gelatin (198μL)×10⁷ dilution: 10⁶-fold diluted phage solution (10 μL)+TBS/gelatin (90μL)×10⁸ dilution: 10⁶-fold diluted phage solution (2 μL)+TBS/gelatin (198μL)×10⁹ dilution: 10⁸-fold diluted phage solution (10 μL)+TBS/gelatin (90μL)

The phage titer was calculated from the following equation.

Titer/mL=colony×1,020 (μL)/200 (μL)×1,000 (μL)/10 (μL)×dilution factor

The total titer of the recovered phages was calculated by multiplyingthe above value by the entire volume (mL) of the recovered phagesolution.

Since the phages employed for reaction have a titer of 6.2×10¹⁰,recovery rate (%) is defined as “total titer of recoveredphages/6.2×10¹⁰”×100.

(5) Screening (Biopanning) for Phage Clones which are Bound to Anti-GD3Antibody

In screening (biopanning) for phage clones expressing peptides capableof binding to GD3 antibody 4F6 with specificity, for the purpose ofefficient screening for phages capable of binding to the Fab region ofGD3 antibody 4F6, GD3 antibody 4F6 was reacted with the phage libraryfrom which phages that bind themselves to standard mouse IgG and/orrecombinant protein A Sepharose had been excluded in advance.

Specifically, biopanning was performed through the following procedure.

Round 1:

Standard mouse IgG-recombinant protein A Sepharose (10 μL) andrecombinant A Sepharose (50 μL) were dissolved in PBS (340 μL), and aphage library (10 μL) (6.2×10¹⁰ titer) was added thereto. The mixturewas allowed to react in a 500-μL Eppendorf tube at 4° C. overnight,followed by centrifugation at 30,000 rpm for 3 minutes, to therebyremove phage particles bound to standard mouse IgG and/or recombinantprotein A Sepharose.

The supernatant (380 μL) and the GD3 antibody 4F6-recombinant protein ASepharose (50 μL) prepared in step (2) above were transferred to a500-μL Eppendorf tube and allowed to react at 4° C. for 5 hours,followed by centrifugation at 3,000 rpm for 3 minutes.

The supernatant was discarded, and PBS (0.5 mL) was added to thesediment, to thereby prepare a suspension. The suspension was thentransferred to a 1.5-mL Eppendorf tube, followed by centrifugation at3,000 rpm for 3 minutes. After this procedure was repeated twice, thesupernatant was discarded, and PBS (500 μL) was added to the sediment,whereby a suspension was obtained. The suspension was transferred to a500-μL Eppendorf tube, followed by centrifugation at 3,000 rpm for 3minutes. The supernatant was discarded, an extraction buffer (50 μL) wasadded to the sediment, and the mixture was left to stand at roomtemperature for 15 minutes under gentle stirring performed every 3minutes, followed by centrifugation at 3,000 rpm for 3 minutes. Thesupernatant was transferred to a concentrator (Centricon™ 30Concentrator: product of Amicon Corporation) and neutralized with 1MTris (pH 9.1) (75 μL), and then TBS (2 mL) was added thereto.

The mixture was centrifuged at 5,000 rpm for 20 minutes, TBS (2 mL) wasadded, and centrifugation was performed at 5,000 rpm for 20 minutes.

The phage solution remaining on the filter of the Centricon wastransferred to a 1.5-mL Eppendorf tube. The filter was washed with TBS(50 μL), and the washings were added to the Eppendorf tube (totalvolume: 470 μL). The obtained phages were amplified (4).

Round 2:

The amplified phages (100 μL) prepared in Round 1 and standard mouseIgG-recombinant protein A Sepharose (10 μL; 25 μg) were dissolved in PBS(350 μL), and the mixture was transferred to a 500-μL Eppendorf tube forreaction at 4° C. overnight. Subsequently, recombinant protein ASepharose (50 μL) was added, and the mixture was allowed to react at 4°C. overnight and then centrifuged at 3,000 rpm for 3 minutes, yielding asupernatant (500 μL).

The supernatant (500 μL) was transferred to a 1.5-mL Eppendorf tube, ananti-GD3 antibody solution (1 mL) was added thereto, and the mixture wasallowed to react at 4° C. overnight, followed by centrifugal separationat 3,000 rpm for 3 minutes.

Subsequently, recombinant protein A Sepharose (50 μL) was added thereto,and the mixture was allowed to react at 4° C. for 3 hours, thencentrifuged at 3,000 rpm for 3 minutes. The supernatant was discarded,and TBS (1 mL) was added to the sediment, to thereby prepare asuspension. The suspension was transferred to a 1.5-mL Eppendorf tubeand then centrifuged at 3,000 rpm for 3 minutes. After this procedurewas repeated twice, the supernatant was discarded, an extraction buffer(50 μL) was added to the sediment, and the mixture was left to stand atroom temperature for 15 minutes under gentle stirring performed every 3minutes, followed by centrifugation at 3,000 rpm for 3 minutes. Thesupernatant was transferred to a concentrator and neutralized with 1MTris (pH 9.1) (75 μL), and then TBS (2 mL) was added thereto. Themixture was centrifuged at 5,000 rpm for 20 minutes, TBS (2 mL) wasadded, and centrifugation was performed at 5,000 rpm for 20 minutes. Thephage solution remaining on the filter of the Centricon was transferredto a 1.5-mL Eppendorf tube. The filter was washed with TBS (50 μL), andthe washings were added to the Eppendorf tube (total volume: 230 μL).The obtained phages were amplified.

Round 3:

The general procedure employed in Round 1 was repeated by use of theamplified phages (100 μL) prepared in Round 2. Thus, through reaction ofphages with an anti-GD3 antibody solution, amplified phages wereobtained. The thus-obtained phages were centrifuged by use of Centricon,whereby eluate was recovered in a total amount of 110 μL.

The results of the above 3 rounds of panning (phage clone recovery ratesof the respective rounds) are shown in Table 1.

TABLE 1 Titer/well fd wild type GD3R-1 GD3R-2 GD3R-3 GD3R-4 10⁹ 0.0240.017 0.055 0.05 0.109 10⁹ 0.08 0.026 0.034 0.086 0.116 10¹⁰ 0.045 0.0670.128 0.058 0.14 10¹⁰ 0.044 0.055 0.109 0.017 0.102 10¹¹ 0.03 0.0940.049 0.094 0.386 10¹¹ 0.029 0.086 0.012 0.108 0.315

In Table 1, “fd wild type” refers to the wild phage strain ofprototype—no peptide being inserted—which was provided to constructphage libraries in Example 1. The prototype phages were prepared throughthe following process and employed in the present test. Briefly, phagevectors formed of DNA molecules of the phages that constitute the phagepeptide library were cleaved for removal of peptide insertion sites, andthe remaining vector fragments were ligated. The thus-reconstructedvectors were used to transform E. Coli JM109 (purchased from Takara),the resultant transformants were cultured with an NZY medium overnight,and the amplified phages were recovered.

Sequencing of the peptides expressed by the phage clones preparedthrough the 3 rounds of panning described above was performed asfollows.

From each of the plates obtained through the above-described titermeasurement performed after completion of 3 rounds of biopanning,arbitrary 32 colonies were randomly picked up, inoculated into a new NZYplate, and incubated at 37° C. overnight. The resultant plate, whichserved as a master plate, was stored at 4° C.

In an NZY medium (20 mL, containing 20 μg/ml TC) placed in a 50-mLcentrifuge tube, each colony of the master plate was suspended, followedby shaking culture at 200 rpm at 37° C. overnight.

The culture was subjected to centrifugal separation at 3,000 rpm for 10minutes. The supernatant was transferred to an Oakridge centrifuge tubeand centrifuged at 12,000 rpm for 10 minutes, to thereby remove E. colicells. The supernatant was transferred to another Oakridge centrifugetube, and PEG6000/NaCl (3 mL; PEG=polyethylene glycol) was addedthereto, followed by thorough stirring. The resultant mixture was leftto stand at 4° C. for 4 hours.

Subsequently, centrifugation was performed at 12,000 rpm for 10 minutesfor causing sedimentation of the phages. The supernatant was removed,and the phage sediment was suspended in TBS (1 mL). The suspension wastransferred to a 1.5-mL Eppendorf tube and centrifuged at 15,000 rpm for10 minutes, to thereby remove insoluble substances. The supernatant wastransferred to another Eppendorf tube, PEG/NaCl (150 μL) was addedthereto, and the mixture was thoroughly stirred and left to stand at 4°C. for 1 hour.

The mixture was centrifuged at 15,000 rpm for 10 minutes, to therebyallow the phages to precipitate. The supernatant was removed, and theprecipitated phage pellet was resuspended in TBS (200 μL). Thesuspension was centrifuged at 15,000 rpm for 10 minutes, to therebysettle insoluble substances. The sediment was transferred to a 0.5-mLEppendorf tube for storage of the phage clones at 4° C.

Extraction of DNA from the thus-obtained phage clones was performed inthe following manner. Phage clones, TBS, and TE-saturated phenol(produced by Nippon Gene Co., Ltd.) were placed in a 1.5-mL Eppendorftube in amounts of 100 μL, 100 μL, and 200 μL, respectively, and themixture was violently stirred for 10 minutes, then subjected tocentrifugal separation at 15,000 rpm for 10 minutes. Subsequently,TE-saturated phenol (200 μL) and chloroform (200 μL) were added to thesupernatant (aqueous phase; 200 μL), and the mixture was violentlystirred for 10 minutes, followed by centrifugal separation at 15,000 rpmfor 10 minutes. TE (250 μL), 3M sodium acetate (40 μL), 20 mg/mLglycogen (product of Boehringer Mannheim; 1 μL), and ethanol (1 ml) wereadded to the supernatant (aqueous phase; 150 μL), and the mixture wasallowed to stand at −20° C. for one hour in a 1.5-mL Eppendorf tube,followed by centrifugal separation at 15,000 rpm for 10 minutes. Thesupernatant was discarded, 80% ethanol (−20° C.; 1 mL) was gently addedto the residue, and the remaining salts were removed through centrifugalseparation at 15,000 rpm for 10 minutes. The supernatant was discarded,moisture within the tube was evaporated, and the DNA that precipitatedwas dissolved in sterilized distilled water (10 μL) for storage at 4° C.

The thus-obtained phage DNAs were subjected to amino acid sequencing ofpeptides.

(6) Amino Acid Sequencing of Selected Peptides

The amino acid sequence of a peptide encoded by phage DNA was determinedby the dye terminator method using a DNA sequence kit (produced byPerkin Elmer, Code; 402079, Lot; A6L015) in accordance with the manualappended to the kit. The DNA sequence of the primer, shown as SEQ ID NO:10, was synthesized with an automatic DNA synthesizer.

Elongation reaction for DNA was performed by use of a thermal cycler(Model 9600, Perkin Elmer, 25 cycles, one cycle consisting of 96° C. for10 seconds, 50° C. for 5 seconds, and 60° C. for 4 minutes). Forsequencing of DNA, a DNA sequencer (ABIPRISM™ 377, product of ABI) wasemployed.

Of the 32 clones, the DNA sequences of 27 clones were successfullydetermined and classified into 4 types. These 4 types of peptides, takenas GD3-mimetic peptides, were named “GD3R1,” “GD3R2,” “GD3R3,” and“GD3R4 (from the highest to the lowest occurrences).

The amino acid sequences of the thus-determined four species ofGD3-mimetic peptides are represented by SEQ ID NOs: 1, 2, 3, and 4, andthe DNA sequences coding for these 4 species of amino acids arerepresented by SEQ ID NOs: 5, 6, 7, and 8.

Example 2 Binding Affinity of GD3-Mimetic Peptide and Anti-GD3 Antibody(ELISA)

A 0.1M NaHCO₃ solution containing each of the phage clones obtained inExample 1 (10¹¹, 10¹⁰, or 10⁹ titer/100 μL) was added to the wells of a96-well microtiter plate (product of Nunc), for immobilization of thephage clones at room temperature for one hour. The supernatant wasremoved, then the wells were subjected to blocking by use of a blockingsolution (400 mL; TBS containing 1% BSA, 0.1% skim milk, and 0.02% Tween20; pH 7.5) at 37° C. for four hours.

The supernatant was removed, then GD3 antibody 4F6 (100 μL), serving asa primary antibody, was added to each well for reaction under shaking atroom temperature for two hours. After completion of reaction, thesupernatant was removed, then the wells were washed six times with awashing solution (400 μL for each well, TBS containing 0.05% Tween 20).Subsequently, a secondary antibody (anti-mouse IgG-HRP, product of SantaCruz Biotechnology, catalogue No. SC-2031, Lot. No. C089) which wasdiluted 5,000-fold by use of a blocking solution prepared in advance wasadded to the wells (100 μL to each well) for reaction under shaking atroom temperature for one hour. After completion of reaction, the wellswere washed four times with a washing solution (400 μL for each well),then a detection reagent (TMB Microwell; product of KPL, Catalogue No.50-76-04, Lot. No. WF075) was added to the wells (100 μL to each well),and the mixture was allowed to stand for five minutes at roomtemperature.

1N-Hydrochloric acid (100 μL) was added to each well for stopping thereaction. Absorbance of each well was measured at 450 nm and 620 nm, and“OD450-OD620” was calculated. A Multiscan (product of Labosystems) wasused in measurement of absorbance. A well in which phages had not beenimmobilized served as a blank, and the absorbance of each well wasdetermined by subtracting the blank value from the “as-measured” value.

The results are shown in FIG. 1.

FIG. 1 shows that GD3R4 exhibits the strongest binding affinity to GD3antibody 4F6.

Example 3 Synthesis of GD3-Mimetic Peptide and Binding Affinity toAnti-GD3 Antibody (1) Synthesis of GD3-Mimetic Peptide

The four species of GD3-mimetic peptide obtained in Example 1 weresynthesized through the following method.

Briefly, by use of an automatic peptide synthesizer (ACT357, product ofAdvanced Chemtech) along with software of the same company, solid-phasesynthesis of respective peptides was performed through the Fmoc/NMP,HOBt method (Fmoc: 9-fluorenylmethoxycarbonyl, NMP: N-methylpyrrolidone,HOBt: 1-hydroxybenzotriazole).

In accordance with the synthesis program, C-terminus-free (OH) peptideswere engineered with reference to the amino acid sequences of SEQ IDNOS: 1-4. Specifically, to 0.25 mmol of “Fmoc-amino acid-Alko resin,”which corresponded to the C-terminus amino acid of the peptide ofinterest, Fmoc-amino acids corresponding to the second amino acid (tothe C-terminus) and subsequent amino acids were sequentially added forelongation of the chain.

Also, peptides each having a C-terminus amide were engineered asfollows. In accordance with the synthesis program, “Fmoc-amino acid,”which corresponded to the C-terminus amino acid of the peptide ofinterest, was added to 0.25 mmol of “Fmoc-NH-SAL resin,” to therebyinduce condensation reaction therebetween, and subsequently, Fmoc-aminoacids corresponding to the second amino acid (to the C-terminus) andsubsequent amino acids were sequentially caused to be attached throughcondensation reaction for elongation of the chain.

After completion of reaction, the N-terminus Fmoc group was deprotectedin accordance with the program.

Each of the thus-obtained peptide resins was collected in a minicolumn(product of Assist) made of polypropylene, followed by washing withmethanol and drying in vacuum. Through the following procedure, thepeptide was cleaved from the resin, and protective groups for the sidechains of the peptide were also removed. Briefly, Reagent K (82.5% TFA,5% phenol, 5% H₂O, 5% thioanisole, and 2.5% ethanedithiol) (2 mL) wasadded to the resin, and reaction was allowed to proceed in theminicolumn for 60 minutes.

Subsequently, the reaction mixture was added dropwise to cold diethylether (8 mL), to thereby stop the reaction and allow the peptide toprecipitate. Thereafter, the mixture in the minicolumn was washed withTFA (2 mL), cold diethyl ether (5 mL) was added to the mixture, themixture was centrifuged, and the precipitated pellet was washed withdiethyl ether (10 mL) four times. Then the peptide was solubilized with50% acetonitrile (about 5 mL) and freeze-dried. This procedure(solubilization and freeze-dring) was repeated twice, whereby a crudefreeze-dried product of interest was obtained.

The freeze-dried product was fractionated by means of reversed-phasehigh-performance liquid chromatography (HPLC) employing an OctadecylColumn (20 (diameter)×250 mm, product of YMC), to thereby isolate thepeptide of interest.

The resins and the amino acid derivative which were used in theabove-described procedure are products of Watanabe.

The thus-isolated respective peptides were identified through amino acidsequencing and molecular weight measurement through mass spectrometry.

(2) Synthesis of Multi-Antigen Peptide

Multi-antigen peptides of the above-obtained GD3-mimetic peptides (MAPs)were synthesized by use of an Fmoc-MAP-Alko resin (product of Watanabe).

Reaction between the Fmoc-MAP-Alko resin (Fmoc₈-Lys₄-Lys₂-Lys-βAla-Alkoresin) and each of the GD3-mimetic peptides proceeded in the same manneras the above-described solid-phase synthesis method.

The structures of the thus-obtained MAPs, shown by one letterrepresentation of amino acid residues, are as follows.

MAP of SEQ ID NO: 1 peptide: (LAPPRPRSELVFLSV)₈-Lys₄-Lys₂-Lys-βAla MAPof SEQ ID NO: 2 peptide: (PHFDSLLYPCELLGC)₈-Lys₄-Lys₂-Lys-βAla MAP ofSEQ ID NO: 3 peptide: (GLAPPDYAERFELLS)₈-Lys₄-Lys₂-Lys-βAla MAP of SEQID NO: 4 peptide: (RHAYRSMAEWGFLYS)₈-Lys₄-Lys₂-Lys-βAla

(3) Binding Affinity to Anti-GD3 Antibody

A 0.1M NaHCO₃ solution containing a GD3-mimetic peptide of the inventionin the above MAP form (100 ng/100 μL) was added to the wells of a96-well micro-titer plate, for immobilization at room temperature forone hour. Subsequently, in a manner similar to the ELISA method employedin Example 2, binding affinity of the GD3-mimetic peptide to GD3antibody 4F6 was investigated.

Anti-GD2 antibody and anti-OAcGD3 antibody (Cerato, E., et al.,Hybridoma, 16(4), 307-316 (1997)) were used as controls (theseantibodies were kindly provided by from Dr. Portoukalian).

The results are shown in Table 2 below.

TABLE 2 Serum % 100% 50% 25% 12.50% Anti-GD3 antibody GD3R1 0.263 0.1370.127 0.083 GD3R2 0.264 0.159 0.105 0.055 GD3R3 0.483 0.357 0.237 0.176GD3R4 0.381 0.309 0.229 0.144 Anti-GD2 antibody GD2R1 0.123 0.095 0.0670.065 GD2R2 0.132 0.079 0.071 0.052 GD2R3 0.191 0.036 0.118 0.077 GD2R40.149 0.103 0.072 0.061 Anti-OAcGD3 antibody GD3R1 0.053 0.046 0.0410.04 GD3R2 0.038 0.027 0.023 0.021 GD3R3 0.044 0.031 0.026 0.031 GD3R40.061 0.047 0.043 0.045

As is apparent from Table 2, among other mimetic peptides, GD3R3 andGD3R4 exhibit stronger bonding to the anti-GD3 antibody. In addition,all the peptides were found to bind to the anti-GD3 antibody strongerthan to the control antibodies.

(4) Synthesis of Fusion Peptide

Fusion peptides—fused with KLH—were prepared by use of each of theabove-obtained GD3-mimetic peptides or an MAP thereof.

Briefly, each of the above-synthesized peptides, or an MAP thereof, andKLH were added to a PBS solution (pH 7.4) containing 0.25%glutaraldehyde at a ratio by weight of 1:10, and the mixture was allowedto react overnight at room temperature, to thereby synthesize a fusionpeptide.

Example 4 Immunization by Use of GD3-Mimetic Peptide (1) Immunization:

Four species of GD3-mimetic peptide (MAP) obtained in Example 3 (2) wasdissolved in PBS so as to attain a concentration of 200 μg/mL. Thesolution was added to Freund's complete (or incomplete) adjuvant (1:1,by volume), to thereby prepare an emulsion.

Eight mice (C57BL/6) were immunized by subcutaneous administration ofthe above emulsion (0.2 mL/mouse, in one administration, 5 μg peptideper mouse). Administration was performed every two weeks (for the secondand subsequent administrations, Freund's incomplete adjuvant was used).After one week following respective administrations, blood was collectedfrom the tail of each mouse, to thereby yield antiserum.

The antiserum obtained directly after the first administration will bereferred to “1-st,” that obtained after the second administration willbe referred to “2-nd,” and that obtained after the third administrationwill be referred to “3-rd.”

(2) Titer Measurement of Antiserum (ELISA):

The titer, against GD3, of each of the antiserum samples (3×8 mice=24samples) obtained in the (1) above was measured through ELISA asdescribed below.

GD3 employed in the test was kindly provided by Dr. Portoukalian, andwas a product extracted from melanoma and purified (J. Portoukalian etal., Int. Cancer, 49, 893-899 (1991)). GD3 was purified by means of HPLCemploying a silica gel (Si60) column (product of Merck, U.S.A.) in achromatogram (Hitachi L-6200). GD3 that had adsorbed onto the columnwall was eluted with a mixture of isopropanol/hexane/purified water(with a gradient from 55/35/12 to 55/30/15 by volume). The flow rate inthe column was 4 mL per minute.

The thus-obtained purified GD3 was dissolved in methanol, whereby a GD3solution having a concentration of 10 μg/mL was prepared. The GD3solution was added to the wells of a 96-well micro-titer plate inamounts of 10 μL per well (which corresponds to 100 ng GD3 per well),and methanol was evaporated. Subsequently, a blocking solution (TBSsupplemented with 1% BSA) was added to the wells in amounts of 50 μL perwell, for blocking at 37° C. for 4 hours.

The supernatant was discarded. Each of the antiserum samples obtained in(1) above was diluted 100-fold, 400-fold, or 1,600 fold with theabove-mentioned blocking solution. The diluted antiserum (50 μL) wasadded to each well for reaction at 4° C. overnight. The well was washedsix times with TBS, and thereafter, HRP-labeled mouse IgG antibodydiluted 5,000-fold with the blocking solution was added (50 μL per well)for reaction for 2 hours at room temperature. The well was washed fourtimes with TBS, and thereafter, enzymatic activity (peroxidase) of thewell was detected with TMB solution (50 μL). The reaction was stoppedwith 1N HCl (50 μL), and the value corresponding to “420 nm-620 nm” wascalculated.

A control sample was prepared as follows. Firstly, an irrelevant peptide(i.e., a peptide of 15 amino acid residues which is different from anyof the four peptides obtained in Example 1 and which does not havebinding affinity with anti-GD3 antibody; the sequence is represented bySEQ ID NO: 10) was synthesized in a manner similar to that employed inExample 3. The synthesized peptide was administered to a mouse forimmunization, and the obtained antiserum served as a control sample. Thetiter of the control sample was obtained in a manner similar to thatdescribed above. (However, the number of provided mice was five, andonly a 100-fold diluted solution was used.) This control peptide samplewas also used in the form of MAP as described above.

Reactivity data of respective antiserum samples with GD3 are summarizedin Tables 3 to 5 below.

TABLE 3 GD3R-1 GD3R-2 Sample Dilution factor Sample Dilution factor No.1/100 1/400 1/1600 No. 1/100 1/400 1/1600 1-1st 0.034 0.017 0.004 1-1st0.316 0.252 0.179 2nd 0.082 0.026 0.01 2nd 0.34 0.241 0.13 3rd 0.0230.01 0.003 3rd 0.113 0.099 0.036 2-1st 0.052 0.039 0.007 2-1st 0.0620.035 0.022 2nd 0.087 0.027 0.016 2nd 0.183 0.078 0.039 3rd 0.068 0.0190 3rd 0.08 0.075 0.023 3-1st 0.047 0.026 0 3-1st 0.042 0.031 0.053 2nd0.14 0.063 0.022 2nd 0.135 0.049 0.035 3rd 0.066 0.017 0.002 3rd 0.0530.053 0.014 4-1st 0.106 0.057 0.005 4-1st 0.114 0.068 0.028 2nd 0.1880.088 0.034 2nd 0.142 0.071 0.025 3rd 0.084 0.03 0.008 3rd 0.057 0.0840.008 5-1st 0.03 0.024 0 5-1st 0.087 0.05 0.045 2nd 0.102 0.074 0.0192nd 0.131 0.034 0.016 3rd 0.139 0.054 0.003 3rd 0.184 0.161 0.053 6-1st0.055 0.052 0.008 6-1st 0.155 0.078 0.028 2nd 0.117 0.047 0.019 2nd0.115 0.091 0.033 3rd 0.084 0.021 0.001 3rd 0.09 0.089 0.028 7-1st 0.0590.034 0 7-1st 0.076 0.055 0.014 2nd 0.168 0.06 0.014 2nd 0.072 0.0390.016 3rd 0.121 0.051 0.004 3rd 0.05 0.059 0.012 8-1st 0.061 0.027 0.0168-1st 0.084 0.044 0.034 2nd 0.054 0.039 0.024 2nd 0.069 0.047 0.034 3rd0.08 0.03 0 3rd 0.098 0.086 0.023

TABLE 4 GD3R-3 GD3R-4 Sample Dilution factor Sample Dilution factor No.1/100 1/400 1/1600 No. 1/100 1/400 1/1600 1-1st 0.062 0.071 0.015 1-1st0.086 0.032 0.007 2nd 0.08 0.054 0.024 2nd 0.122 0.058 0.019 3rd 0.0470.022 0.016 3rd 0.116 0.053 0.013 2-1st 0.169 0.083 0.017 2-1st 0.0420.021 0.014 2nd 0.116 0.078 0.025 2nd 0.08 0.07 0.022 3rd 0.126 0.0390.027 3rd 0.088 0.042 0.008 3-1st 0.105 0.077 0.009 3-1st 0.071 0.0470.014 2nd 0.098 0.079 0.025 2nd 0.164 0.114 0.026 3rd 0.143 0.032 0.0263rd 0.176 0.093 0.029 4-1st 0.062 0.004 0.016 4-1st 0.247 0.108 0.0522nd 0.08 0.112 0.034 2nd 0.195 0.128 0.062 3rd 0.069 0.028 0.018 3rd0.253 0.111 0.026 5-1st 0.098 0.048 0.006 5-1st 0.086 0.033 0.009 2nd0.165 0.118 0.022 2nd 0.153 0.093 0.033 3rd 0.189 0.073 0.017 3rd 0.190.104 0.022 6-1st 0.051 0.016 0.023 6-1st 0.08 0.041 0.008 2nd 0.1320.088 0.027 2nd 0.124 0.106 0.031 3rd 0.189 0.033 0.018 3rd 0.168 0.0980.039 7-1st 0.04 0.026 0.003 7-1st 0.063 0.034 0.006 2nd 0.135 0.0760.018 2nd 0.147 0.079 0.041 3rd 0.073 0.03 0.013 3rd 0.113 0.045 0.0138-1st 0.165 0.067 0.031 8-1st 0.082 0.037 0.012 2nd 0.179 0.088 0.07 2nd0.05 0.057 0.013 3rd 0.098 0.023 0.018 3rd 0.147 0.061 0.024

TABLE 5 Control peptide Sample No. Dilu. factor 1/100 1-1st 0.105 2nd0.064 3rd 0.006 2-1st 0.088 2nd 0.056 3rd 0 3-1st 0.057 2nd 0.042 3rd0.007 4-1st 0.041 2nd 0.028 3rd 0.004 5-1st 0.051 2nd 0.031 3rd 0.031

As is apparent from form Tables 3 to 5, the antiserum samples obtainedthrough use, as an immunogen, of a GD3-mimetic peptide of the presentinvention cross-react with GD3; in particular, those obtained throughuse, as an immunogen, of a GD3R4 of the present invention exhibit highreactivity with GD3. In contrast, antiserum samples obtained through useof the control peptide was found to exhibit weak reactivity with GD3,and taken together, antisera obtained through use of a GD3-mimeticpeptide of the present invention are suggested to cross-react with GD3.

The above results suggest that GD3-mimetic peptides of the presentinvention, in particular GD3R4, mimic a portion of the structure of GD3;i.e., a structural portion recognized by anti-GD3 antibody 4F6.

Example 5 Reactivity of GD3-Mimetic Peptide

(1) In a manner similar to that described in Example 3, peptides ofN-terminus 9 amino acid residues or C-terminus 9 amino acid residues ofGD3R1, GD3R2, GD3R3, or GD3R4. The nomenclature and sequences ofrespective peptides employed in the present test are shown in FIG. 2These were employed as multi-antigen peptides (MAPs).(2) In order to extend the research on bonding between the anti-GD3antibody 4F6 and each of the GD3-mimetic peptides shown in FIG. 1,multi-antigen peptides (MAPs) were subjected to ELISA. MAP sampleshaving a variety of concentrations shown on the X-axis in FIG. 3 wereprovided, and each sample was immobilized onto the wells of a 96-wellplate. Subsequently, the wells were blocked with a blocking solution(TBS supplemented with 1% BSA, 0.1% skim milk, and 0.02% Tween 20) at 4°C. overnight. 4F6 (hybridoma supernatant: used in the neat form) wasadded (100 μL/well) for reaction at room temperature for 2 hours. Aftercompletion of reaction, the supernatant was discarded, and the wellswere washed with a washing solution (TBS supplemented with 1% FBS and0.05% Tween 20) six times. Subsequently, the wells were allowed to reactwith peroxidase-labeled anti-mouse IgG (diluted 1,000-fold with ablocking solution) at room temperature for 2 hours. The wells were againwashed with a washing solution four times, and the amount of peroxidaseenzyme remaining in each well was detected and quantitatively determinedwith the substrate TMB, whereby binding of MAP to 4F6 was investigated.The test was performed in triplicate, and the values in the graph are“mean ±SD.”

As understood from FIG. 3, 4F6 binds to GD3-mimetic peptides 3 or 4 inan amount-dependent manner. Although bonding to GD3 appears to havedeclined from the concentration 0.8 μg/well and higher, this may beattributed to excessive amount of immobilized GD3 present on the ELISAplate, which intensified hydrophobicity, thereby inhibiting binding tothe antibody.

(3) The binding affinity shown in FIG. 3 was investigated in anothermethod. Briefly, MAP samples having a variety of concentrations shown onthe X-axis in FIG. 4 were provided. Each sample was immobilized onto thewells of a 96-well plate, and the wells were allowed to react with 4F6(100 μL/well) at 4° C. overnight. On the following day, the supernatant(80 μL) was collected and added to the wells of another plate to whichGD3 had been immobilized in advance (100 ng/well) for reaction at roomtemperature for 2 hours. After completion of reaction, the amount ofantibody 4F6 that had been condensed with GD3 (i.e., that had not beenabsorbed by MAP) was detected and quantitatively determined through themethod described (2) above, whereby inhibitory effect of MAP on bindingof GD3 to 4F6 was investigated. The test was performed in triplicate,and the values in the graph are “mean ±SD.”

As a result, as shown in FIG. 4, like the case of GD3, both theGD3-mimetic peptides 3 and 4 were found to inhibit GD3 from binding tothe antibody, and the inhibition occurred in a manner dependent on theamount immobilized.

(4) Next, investigation was made as to whether binding between GD3R3 orGD3R4 and 4F6 occurred at the GD3 binding site of 4F6. To the wells of aplate to which GD3 had been immobilized (100 ng/well), each of the MAPsamples having a variety of concentrations shown on the X-axis in FIG. 5and 4F6 (100 μL/well) were simultaneously added, whereby an inhibitorytest was performed. The test was performed in triplicate, and the valuesin the graph are “mean ±SD.”

As a result, as is apparent from FIG. 5, within the concentration rangeemployed in the present test, GD3R3, like GD3, exhibited inhibitoryeffect, but R4 did not. From these results, the following two arededuced. Firstly, the binding strength between 4F6 and respective MAPsdecreases in the order of GD3>GD3R3>GD3R4. Secondly, GD3R3 is bound tothe GD3 binding site, or in the vicinity, of 4F6.

(5) Through the procedure described above, among other mimetic peptides,GD3R3 has been found to specifically bind to the GD3-binding domain of4F6 or a site close thereto. Next, in an attempt to determine the domainrequired for establishing binding to anti-GD3 antibody 4F6 inGD3-mimetic peptides, an MAP of nine N-terminus residues of GD3R3peptide and an MAP of nine C-terminus residues of the same GD3R3 peptideas shown in FIG. 2 were synthesized, their binding to 4F6 was studied bymeans of ELISA. ELISA was performed was performed in triplicate, and thevalues in the graphs are “mean ±SD.”

As a result, as is apparent from FIG. 6, GD3R3C9 was found to havecomparable ability to the 15 residues in inhibiting binding between GD3and 4F6, and the inhibition occurred in a manner dependent on the amountimmobilized.

Taken together, it has become clear that GD3R3 is the strongest inbinding to anti-GD3 antibody 4F6, and that nine residues from theC-terminus (GD3R3C9) is critical in establishing binding.

Example 6 Immunization with Fusion Peptide (1) Immunization

Using a fusion peptide R4-KLH obtained in Example 3 (4) between GD3R4and KLH or a MAP form fusion peptide R4MAP-KLH created between GD3R4 andKLH, immunization was performed as described in (1) above.

Briefly, an emulsion mixed with an adjuvant (1:1, by volume) wasadministered to each member of groups of mice, each group consisting of3 mice (CD-1), in an amount of 100 μL/mouse (for one administration(ip), peptide 30 μg/mouse) for immunization. In the case of R4-KLH,administration was performed every one week for one month, andsubsequently every two weeks for one month; and in the case ofR4MAP-KLH, every one week for 2 months. In any case, 4 days after thefinal immunization, blood was collected to thereby prepare antiserumsamples.

Similar to the case of immunization with R4-KLH, antiserum samples werealso prepared through immunization with GD3 (30 μg).

(2) ELISA Test

Through use of an ELISA plate to which GD3 or GD3R4 peptide had beenimmobilized, the procedure described in Example 4 (2) was repeated.

GD3-Immobilized plate: GD3 in methanol-PBS (1:1) (0.5 μg/50 μL) wasadded to each well, and the plate was left to stand for one hour. Thewells were washed with PBS, then 1% HSA in PBS was added thereto (200μL/well) for incubation at 37° C. for 2 hours, whereby the wells wereblocked.

GD3R4 Peptide immobilized plate: GD3R4 peptide (1 μg) dissolved in 0.1Mbicarbonate buffer (pH 9.5) was added to each well, followed byincubation at 37° C. overnight and washing with PBS, whereby the wellswere blocked in a manner similar to the above.

The antiserum samples obtained in the above were diluted with PBS tothereby yield 100-fold to 10,000-fold diluted antiserum samples. Each ofthe resultant diluted antiserum samples (50 μL) was added to each welland the mixture was allowed to react for one hour. Subsequently,reaction was allowed to proceed with biotinylated anti-mouseimmunoglobulin antibody (Ig, IgM, IgG, IgG1, IgG2a, IgG2b or IgG3specific antibody) for one hour, then with streptoavidin-HRP for onehour in a similar manner, after which enzymatic activity in each wellwas detected as described above (405 nm).

(3) Cell Response Test

The spleen aseptically removed from each of the above-immunized mice wasminced in a 10% FCS-supplemented RPMI 1640 medium. By use of a nylonwool column, T-cell-rich lymphocytes were prepared and the cells werecounted. The above medium containing the cells in an amount of 10⁵ cellsper 150 μL was added to each of the wells of a culture plate. PHA wasadded so as to attain a final concentration of 1 μg/mL. A peptide of thepresent invention or any of different gangliosides in PBS was added, andthe mixture was incubated for 96 hours. A supernatant (100 μL) wasobtained from each well. The IL-2 activity of the supernatant wasdetermined through use of IL-2-dependent mouse cells, CTLL2. Briefly,the supernatant (100 μL) diluted 2 to 50-fold with a medium was added tothe wells in an amount of 10⁴ CTLL2 cells/well, followed by incubationat 37° C. for 48 hours. Subsequently, 3H-thymidine (0.5 μCi) was addedto each well, followed by incubation for 6 hours. CTLL2 cells wererecovered on a paper filter and counted for 3H.

(4) Results

Antiserum samples obtained from immunization with either R4-KLH orR4MAP-KLH were found to contain specific antibodies that react with boththe GD3R4 peptide and GD3.

The titers of IgG antibodies and that of IgM antibodies were almost thesame. The results regarding reactivity between GD3 and antiserumobtained from immunization with R4-KLH are shown in FIG. 7. Reactivitybetween GD3 and antiserum obtained from immunization with R4MAP-KLH andreactivity between GD3 and antiserum obtained from immunization withGD3R4 are shown in FIGS. 8 and 9, respectively.

T-cell-rich lymphocytes from GD3-immunized mouse are considered notactivated, because IL-2 activity was not detected in the presence ofgangliosides (Table 6). However, in the presence of GD3R4 peptide orGD3R3 peptide, production of IL-2 has been confirmed (Table 6). In thisconnection, in R4-KLH-immunized mice, T cells have been confirmed tohave activated by GD3R4 peptide or GD3R3 peptide (Table 7). Theseresults indicate that the peptides of the present invention induceactivation of specific T cells in GD3-immunized mice.

TABLE 6 3H Thymidine up-take into IL-R-dependent CTLL2 cells incubatedin culture supernatant of spleen cells of mice immunized with GD3 andstimulated with PHA for 96 hours (in the presence of a variety ofgangliosides or peptides) Stimulation Dilution factor of culturesupernatant (PHA 1 (μg/mL+) ½ ⅕ 1/20 1/50 PHA alone 2631 ± 248 1797 ±169 804 ± 82 183 ± 19 GD3(0.5 μg/ml) 2795 ± 175 1876 ± 152 810 ± 72 207± 22 GD3(1 μg/ml) 2568 ± 235 1834 ± 169 762 ± 55 186 ± 27 GD3(2 μg/ml)2453 ± 264 1901 ± 188 758 ± 61 197 ± 28 GD3(5 μg/ml) 2656 ± 257 1857 ±185 794 ± 68 179 ± 23 GM3(0.5 μg/ml) 2567 ± 248 1897 ± 190 824 ± 81 182± 22 GM3(1 μg/ml) 2792 ± 218 1728 ± 157 833 ± 90 178 ± 24 GM3(2 μg/ml)2725 ± 232 1824 ± 163 859 ± 82 181 ± 28 GM3(5 μg/ml) 2658 ± 247 1751 ±183 832 ± 81 179 ± 22 GM1(0.5 μg/ml) 2754 ± 282 1841 ± 167 804 ± 84 182± 19 GM1(1 μg/ml) 2649 ± 255 1748 ± 181 825 ± 77 175 ± 27 GM1(2 μg/ml)2842 ± 248 1697 ± 190 783 ± 81 167 ± 21 GM1(5 μg/ml) 2737 ± 264 1754 ±184 756 ± 76 182 ± 26 peptide R4(1 μg/ml)  5867 ± 1213 3418 ± 749 1994 ±348  881 ± 127 peptide R4(2 μg/ml) 12671 ± 1643  8349 ± 1014 3248 ± 6131274 ± 156 peptide R4(5 μg/ml) 14237 ± 1884 11358 ± 1563 7526 ± 839 2684± 387 peptide R1(1 μg/ml) 3014 ± 671 1967 ± 276  888 ± 107 234 ± 43peptide R1(2 μg/ml) 3219 ± 546 1831 ± 259  906 ± 121 246 ± 38 peptideR1(5 μg/ml) 3344 ± 497 1956 ± 285  942 ± 116 251 ± 44 peptide R2(1μg/ml) 2957 ± 326 1784 ± 164 792 ± 88 185 ± 31 peptide R2(2 μg/ml) 3057± 321 1719 ± 184  856 ± 107 192 ± 40 peptide R2(5 μg/ml) 3022 ± 316 1717± 198  931 ± 124 203 ± 36 peptide R3(1 μg/ml) 4066 ± 853 2654 ± 486 1153± 246  571 ± 104 peptide R3(2 μg/ml)  5791 ± 1154 3243 ± 634 1540 ± 467 749 ± 118 peptide R3(5 μg/ml)  7536 ± 1247 3528 ± 543 1566 ± 328  761 ±102 The values are mean ± SE (4 wells)

TABLE 7 3H Thymidine up-take into IL-2-dependent CTLL2 cells incubatedin culture supernatant of spleen cells of mice immunized with R4 peptideand stimulated with PHA for 96 hours (in the presence of a variety ofgangliosides or peptides) Stimulation Dilution factor of culturesupernatant (PHA 1 μg/mL+) ½ ⅕ 1/20 1/50 PHA alone 2732 ± 265 1628 ± 147813 ± 96 164 ± 28 GD3(0.5 μg/ml) 2635 ± 308 1792 ± 154 850 ± 87 169 ± 25GD3(1 μg/ml) 2721 ± 284 1658 ± 134 794 ± 82 202 ± 24 GD3(2 μg/ml) 2797 ±295 1722 ± 163 809 ± 74 212 ± 28 GD3(5 μg/ml) 2619 ± 316 1636 ± 145 764± 68 184 ± 23 GM3(0.5 μg/ml) 2741 ± 279 1683 ± 167 812 ± 84 167 ± 21GM3(1 μg/ml) 2814 ± 321 1728 ± 181 873 ± 90 194 ± 25 GM3(2 μg/ml) 2754 ±286 1619 ± 155 768 ± 83 157 ± 22 GM3(5 μg/ml) 2631 ± 264 1587 ± 163 729± 89 152 ± 26 GM1(0.5 μg/ml) 2750 ± 317 1683 ± 178 801 ± 93 204 ± 25GM1(1 μg/ml) 2812 ± 309 1715 ± 186 826 ± 75 210 ± 31 GM1(2 μg/ml) 2788 ±274 1664 ± 191 837 ± 78 224 ± 29 GM1(5 μg/ml) 2764 ± 285 1728 ± 183 872± 71 219 ± 28 peptide R4(1 μg/ml)  6781 ± 1549 4413 ± 854 2420 ± 5611017 ± 182 peptide R4(2 μg/ml) 15383 ± 2194 10375 ± 1708  7934 ± 10312742 ± 751 peptide R4(5 μg/ml) 18692 ± 2310 12657 ± 1624 8354 ± 957 3058± 568 peptide R1(1 μg/ml) 3218 ± 716 1876 ± 281  949 ± 136 225 ± 38peptide R1(2 μg/ml) 3417 ± 542 1864 ± 327  845 ± 147 220 ± 40 peptideR1(5 μg/ml) 3469 ± 612 1781 ± 285  901 ± 152 236 ± 45 peptide R2(1μg/ml) 2857 ± 493 1714 ± 239  745 ± 136 178 ± 38 peptide R2(2 μg/ml)2764 ± 322 1653 ± 286  694 ± 141 163 ± 41 peptide R2(5 μg/ml) 2849 ± 3961628 ± 245  748 ± 162 174 ± 32 peptide R3(1 μg/ml)  5577 ± 1063 2986 ±327 1491 ± 364  682 ± 120 peptide R3(2 μg/ml)  6351 ± 1314 3184 ± 4391527 ± 337  736 ± 137 peptide R3(5 μg/ml)  7898 ± 1496 3652 ± 681 1684 ±375  812 ± 151 The values are mean ± SE (4 wells)

INDUSTRIAL APPLICABILITY

According to the present invention, there are provided novel amino acidsequences that mimic GD3 expressing on cancerous tissue or cancer cellsurfaces. The GD3-mimetized peptides of the present invention containingany one of the amino acid sequences find utility for the preparation ofdrugs, including diagnostic agents for cancer or cancer vaccines. Thepresent invention provides method for treatment of cancer, method fordiagnosing cancer, etc., thus contributing improvement of therapeuticeffect of cancer therapy.

1. A GD3-mimetic peptide containing an amino acid sequence representedby SEQ ID NO:3 or an amino acid sequence derived therefrom bysubstitution, deletion, addition, or insertion of one or more amino acidresidues and attaining specific binding to an anti-GD3 antibody.
 2. AGD3-mimetic peptide as described in claim 1, which is in a fused form ofpeptide, fused with a carrier protein enhancing immunogenicity.
 3. Afused GD3-mimetic peptide as described in claim 2, wherein the carrierprotein is keyhole limpet hemocyanin.
 4. A GD3-mimetic peptide asdescribed in claim 1, which is in a form of multi-antigen peptidecontaining at least one species of the GD3-mimetic peptide.
 5. AGD3-mimetic peptide as described in claim 1, which is an immunogenicpeptide having the ability to produce a GD3-specific antibody.
 6. AGD3-mimetic peptide as described in claim 1, which is an amino acidsequence represented by SEQ ID NO:3.
 7. A pharmaceutical compositioncontaining, as an active ingredient, a GD3-mimetic peptide as recited inclaim 1.